動物簊因轉殖技術與實驗\(總-送印版\)

2008

: http://www.as.nchu.edu.tw/bio-tech/

.... 1 4... 17 34.... 48 58 RNAi RCAS-TVA... 71... 89... 99... 117... 131... 144... 155... 172 189... 201... 211 219... 239.. 248 -... 264.. 286... 290 DNA.. 297... 303... 307 I

... 312... 318 DNA.. 324 ---Southern blot hybridization... 329 PCR Slot blot 335 RNA.. 341 ---Northern blot hybridization & RT-PCR 348... 362 ---Western blot hybridization. 367......... 372...... 376 II

, (tissue-specific gene expression) 酶 human tissue plasminogen activator, tpa β- 1

promoter 300 kg 10% 2/3 20 kg 1/10 Pharmaceutical Proteins Bayer Antitrypsin DNA DNA 2

2002β- / 31:71 2001 : 30:45-48 Houdebine L. M. 2002. The methods to generate transgenic animals and to control transgene expression. J. Biotechnol. 98:145-160. Houdebine L. M., S. Rival, T. Pantano, G. Jolivet, D. Thepot and J. Attal. 2002. Transgenesis for the study and the control of lactation. Reprod. Nutr. Dev. 42:117-125. Huang, M. C. and J. S. Chao. 2001. Regulatory sequences of the 5' flanking region of goat β-casein gene. AJAS 14:1628-1633. Pintado B. and A. Gutierrez-Adan. 1999. Transgenesis in large domestic species: future development for milk modification. Reprod. Nutr. Dev. 39:535-544. Soukharev S., D. Hammond, N. M. Ananyeva, J. A. Anderson, C. A. Hauser, S. Pipe and E. L. Saenko. 2002. Expression of factor VIII in recombinant and transgenic systems. Blood Cells Mol. Dis. 28:234-248. Takahashi R. and M. Ueda. 2001.The milk protein promoter is a useful tool for developing a rat with tolerance to a human protein. Transgenic Res 10:571-575. Wu, H. T. and M. C. Huang. 1995. Effects of electroporation on the quality of rooster semen. AJAS 8:175-178. 3

1 2 1 2 DNA DNA 4

DNA DEAE electroporation Zhu 1985 Zhu MT-hGH Southern blot hybridization cyprinids: carp salmonids: salmon and trout cichlids: tilapia catfish 80% transgenic mice one cell embryo pronucleous foster mother zebrafish, Danio rerio medaka, Oryzias latipes zebracichlid 5

vector promoter enhancer vector 1. SV40 SV40 DNA DNA 5,200 early late lytic cycle DNA DNA DNA origin of viral DNA replication T large T antigen nonpermissive DNA capsid SV40 SV40 DNA DNA recombinants DNA SV40 DNA DNA DNA plasmid-like SV40 DNA SV40 DNA SV40 DNA COS DNA β- β-globin spliced mrna 2. 6

DNA bovine papilloma virus, BPV DNA 8,000 BPV DNA 30 100 DNA plasmid DNA DNA DNA insulin BPV DNA DNA 30 50 mrna BPV DNA pbr322 3. retrovirus RNA 5 methlated cap 3 A poly-a tail mrna RNA DNA DNA provirus DNA LTR RNA RNA RNA splicing DNA DNA LTR mrna SV40 SV40 T 7

enhancer sequences SV40 72 repeated in a tandem fashion 72 β- β- mrna β- mrna 1. DNA tk tk DNA Ca ++ tk DNA DNA Ca ++ Ca ++ tk DNA carrier Ca ++ DNA DNA 2. DNA 0.1 0.5 µm microinjection micropipette puller micromanipulator DNA 3. DNA DNA 4. DNA DNA lipid bilayer 8

electrical breakdown V 3/2 r E Cosθ 1 V r θ E phospholipid bilayer V 1 1 Cos 0 1 Cos 90 0 Neumann 1992 a DNA E b DNA c DNAQ d e 5. 20 Hommond Whitten 9

1956 37 CO 2 Binster 1980 mrna Solter 1984 ES cell 1981 Martin Evans Kaufman 1984 Bradley The Sanger Institute ES cell ES cell DNARNA DNA DNA DNA 1. DNA 2. DNA 3. DNA DNA 10

DNA 30% 10% 1. 4 2. DNA 3. DNA transfect DNA 1. 2. DNA 3. 4. DNA 5. 11

* * * * * * * * * ***** * * * * DNA (*) ******** a) b) * * ******** Mature and fertilize oocyte in vitro c) *** ** ** **** DNA 12

**** a) * b) * c) DNA Isolate embryonic stem (ES) cells from the inner cell mass cells of a blastocysts Transfect ES cells with foreign DNA Screen ES cell colonies for expression or integration of the Transgene Grow clonal cell lines containing the transgene in vitro Many embryos can be produced carring the transgene in the same location in the genome 13

14

1993 Bioindustry 4:37-46 1994 :29-47 1993 482:39-50 1998 Bioindustry 9:1-9 Boyton, R. J. and D. M. Altmann. 2002. Transgenic models of autoimmune disease. Clin. Exp. Immunol. 127:4-11. Finn, O. J. and G. Forni. 2002. Prophylactic cancer vaccines. Curr. Opin. Immunol. 14:172-177. Greten, F. R., M. Wagner, C. K. Weber, U. Zechner, G. Adler and R. M. Schmid. 2001. TGF alpha transgenic mice: a model of pancreatic cancer development. Pancreatology 1:363-368. Hard, G. 2002. A search for an animal model of the Spanish toxic oil syndrome. Food Chem. Toxicol. 40:1551. Koike, K., T. Tsutsumi, H. Fujie, Y. Shintani and M. Kyoji. 2002. Molecular mechanism of viral hepatocarcinogenesis. Oncology 62:29-37. Kozak, L. P. and M. Rossmeisl. 2002. Adiposity and the development of diabetes in mouse genetic models. Ann. N. Y. Acad. Sci. 967:80-87. Lamartiniere, C. A., M. S. Cotroneo, W. A. Fritz, J. Wang, R. Mentor-Marcel and A. Elgavish. 2002. Genistein chemoprevention: timing and mechanisms of action in murine mammary and prostate. J. Nutr. 132:552S-558S. McClain, D. A. 2002. Hexosamines as mediators of nutrient sensing and regulation in diabetes. J. Diabetes Complications 16:72-80. Olgin, J. E. and S. Verheule. 2002. Transgenic and knockout mouse models of atrial arrhythmias. Cardiovasc. Res. 54:280-286. Szalai, A. J. 2002. The antimicrobial activity of C-reactive protein. Microbes. Infect. 4:201-205. Zaugg, M., M. C. Schaub, T. Pasch and D. R. Spahn. 2002. Modulation of beta-adrenergic receptor subtype activities in perioperative medicine: mechanisms and sites of action. Br. J. 15

Anaesth. 88:101-123. 16

- fertilization primodial germ cells, PGC allantois genital ridge zona pellucida, ZP meiosis haploid DNA 17

homologous chromosomes synapsis 2 n n 20 2 20 DNA polar body mrna 1. leptotene DNA 2. zygotene synaptonemal complex 4 tetrads 18

3. pachytene 4 2 100 nm recombination nodules 4. diplotene X- chiasmata DNA lampbrush chromosome loops RNA DNA 5. diakinesis DNA prometaphase spindle fiber equatarial plate 19

terminization congression sister chromatids interphase polar body interkinesis DNA 20

n gametogenesis ameboid movement hindgut genital ridge primitive sex cords mesonephros testis determining factor, TDF Y 10 1/3 oogenesis oogonia Y celluar clusters primodial follicle 110 2,700,000 21

70,000 primary oocytes dictyate germinal vesicle, GVa first meiotic arrest puberty gonadotropins sex steroids cytokines growth factors atresia primary follicle zona pellucida, ZP secondary follicle antrum antral follicles corona radiate theca cells tertiary follicle Graafian follicles follicle stimulating hormone, FSH luteinizing hormone, LH first polar body secondary oocytes b 22

second meiotic arrest preovulatory follicles LH surge second polar body spermatogenesis spermatogonia spermatid spermiogenesis spermatozoa seminiferons tubules stem cells intercellular bridges Ap A4 A8 A16 A32 32 32 A16 A32 A1 A1 A2 A3 A4 B preleptotene primary 23

spermatocyte sertoli cells epididymis motile sperm 61 65 75 golgi phase cap phase acrosomal phase maturation phase acrosomal granules headtail flagellum acrosome ATP 9+2 9 microtubules central filaments 9 24

fertilization 2 capacitation hyperactivation receptor adenylate cyclase ATP campcamp 2 protein kinase phosphorylation dynein 15 20 µm ZP1 ZP2 ZP3 ZP3 ZP2 ZP1 ZP2 ZP3 25

ZP3 sperm receptor ZP1 exocytosis protein-digesting enzymes hydrolases ion channels Na + membrane potential depolarization 1 3 fast block to polyspermy multiple fertilization follicular cells cortical granules G G protein ZP3 ZP2 cortical reaction activation parthenogenesis fertilin male 26

pronucleus 2 2 2 female pronuclei 2 20 23 syngamy zygote 27 28 cleavage blastomeres cell mass hatch RNA inactive 1 3 2-4- 8- totipotency 16-16- 1997 polarization inncer cell mass, ICM trophectoderm 27

the emergence of pattern change in form 28

( ) ( ) 2n 4n 2C DNA n+n a. b. a. b. 29

(A1) (A2) (A3) (A4) (I) (B) A1 A2 A3 A4 B (primary spermatocyte) (secondary spermatocyte) 30

a. b. c. d. a. b. c. d. 31

a. b. c. d. a. 2- b. 4- c. d. 32

Campell, N. A. and J. B. Reece. 2002. Biology. 6 th ed., pp. 234-246, 980-1011. Benjamin Cummings San Francisco, USA. Garner, D. L. and E. F. E. Hafez. 2000. Spermatozoa and seminal plasma. In: Reproduction in farm animals. 7 th ed., pp. 96-109. Lippincott Williams & Wilkins, Philadelphia, USA. Hafez, E. S. E. and B. Hafez. 2000. Folliculogenesis, egg maturation, and ovulation. In: Reproduction in Farm Animals. 7 th ed., pp. 68-81. Lippincott Williams & Wilkins, Philadelphia, USA. Izadyar, F., L. B. Creemers, F. M. F., van Dissel-Emiliani, A. M. M., van Pelt and D. G., de Rooij. 2000. Spermatogonial stem cell transplantation. Mol. Cell Endocriol. 169:21-26. Karp, G. 1999. Cell and molecular biology: Concepts and Experiment. 2 nd ed., pp. 635-641. John Wiley & Sons, NY, USA. McLean, D. J., D. S. Johnston, L. D. Russell, M. D. Griswold. 2001. Germ cell transplantation and the study of testicular function. Trends Endocrinol. Metab. 12:16-21. Ogawa, T. 2001. Spermatogonial transplantation: the principle and possible applications. J. Mol. Med. 79:368-374. Senger, P. L. 1997. Pathways to pregnancy and parturition. 1 st ed., pp. 62-76, pp. 130-147, pp. 168-186. Current Conceptions, WA, USA. Ventela, S, H. Ohta, M. Parvinen, Y. Nishimune. 2002. Development of the stages of the cycle in mouse seminiferous epithelium after transplantation of green fluorescent protein-labeled spermatogonial stem cells. Biol. Reprod. 66:1422-1429. Wolpert, L., R. Beddington, J. Brockes, T. Jessesll, P. Lawrence and E. Meyerowitz. 1998. Principles of development. pp.1-59. Current Biology Ltd, London. 33

in vitro production, IVP in vitro maturation, IVM in vitro fertilization, IVF in vitro culture, IVC transgenic technology animal cloning folliculogenesis gonadotropins local regulation factors steroids cytokines growth factors primary follicle primary oocytes 2,500/75,000 atretic follicle primordial germ cells, PGC oogonia 34

mitosis mesenchymal cells pregranulosa cells primordial follicle primary follicle zona pellucida, ZP secondary follicle follicular fluid antrum antral follicles stimulating hormone, FSH estrogen receptor FSH corona radiate theca cells theca interna theca externa androgen luteinizing hormone, LH atresia Graffian follicles FSH preovulatory follicles LH LH 1. primary oocytes leptotene zygotene pachytene 35

diplotene prophase I dictyate germinal vesicle, GV first meiotic arrest puberty follicles-stimulating hormone, FSH luteinizing hormone, LH resumption of meiosis germinal vesicle breakdown, GVBD prometaphase I, ProM I metaphase I, MI anaphase I, AI telophase I, TI metaphase II, MII a first polar body nuclear maturation secondary oocytes second meiotic arrest expansion mucification FSH cortical granules 2. LH GVBD 36

1 hormones FSH LH releasing hormone, LH-RH LH PGE2 PGF2α 2 cyclic adenosine-3-5 - monophosphate, camp oocyte maturation inhibitor, OMI camp camp OMI tight junctions camp camp LH OMI OMI OMI 3 maturation promoting factor, MPF cytostatic factor, CSF 1985 Fulka MI GV GVBD MI MPFMPF 34 kd protein kinasep34 cdc2 cyclin B GV GV cyclin B P34 cdc2 GVBD MPF P34 cdc2 cyclin B 37

pre-mpf dephosphorylation MPF GV nuclear envelope breakdown, NEBD MPF 8 16 GVBD MI MPF MPF MII Ca 2+ cyclin B MPF Xenopus CSF c-mos Ca 2+ CSF MPF GVBD CSF Xenopus MII MPF CSF MII MII CSF-Like Xenopus CSF MPF in vitro maturation, IVM Manipulation of oocyte-enclosed preantral follicle cumulus oocytes complexes, COCs IVM GV MII IVM 38

IVM hr M-199 24 M-199 22 26 NCSU 23, M-199 40 44 M-199, F10, F12 24 26 M2, HTF 24 MII cytoplasmic maturation FSH LH 80 90% 40 80% 25 30 38 COCs 1. M-199 2. 35 mm 3 5 100 µl 3 4 ml 2 COCs IVM 5% CO 2 38.5 20 24 39

Component Concentration of stock solution For 20 ml medium FSH 1 mg /ml 10 µl LH 1 mg /ml 100 µl Estradiol 1 mg /ml 40 µl Pyruvate 250 mm 20 µl M-199+7.5% FCS Added to 20 ml ROSI, ICSI, NT GV GV IVM KSOM HTF KSOM Component Concentration, mm g/l NaCl 95.0 5.55 KCl 2.50 0.186 KH 2 PO 4 0.35 0.0476 MgSO 4 0.20 0.0493 Na lactate 10.00 1.87 Glucose 0.20 0.036 Na pyruvate 0.20 0.022 NaHCO 3 25.0 2.10 CaCl 2 2H 2 O 1.71 0.251 Gutamine 1.0 0.146 EDTA 0.01 0.0038 BSA 1.0 mg/ml Penicillin-G 100 U/mL 60U/mL Streptomycin sulfate 50 U/mL Modified from Biggers et al. 2000 40

HTF* Component mm NaCl 101.6 KCl 4.69 MgSO 4 0.2 KH 2 PO 4 0.37 Na lactate 1.32 g/ml 21.4 Glucose 2.78 CaCl 2 2.04 NaHCO 3 25 CaCl 2 2H 2 O Na pyruvate 0.33 Phenol red 10 mg/l Penicillin-G 1597 units/mg 100 units/ml Streptomycin sulfate 50 mg/l *HTF: human tubular fluid attach Ca 2+ 3 6 cortical granule reaction zona reaction Ca 2+ activation Ca 2+ smooth endoplasmic reticulum Ca 2+ 20 25 1 2 19 20 Miyazaki 1990 Ca 2+ Ca 2+ Miyazaki receptor-mediated 41

G-protein phosphatidylinositol 4,5-bisphosphate PIP2 inositol 1,4,5-trisphosphate IP3 DA IP3 second messenger Ca 2+ Ca 2+ Ca 2+ third messenger Ca 2+ Ca 2+ MPF Stice Robl 1990 IP3 Ca 2+ SOAF sperm-borne oocyte-activating factor Ca 2+ parthenogenesis Ca 2+ MII CSF MPF MPF CSF Ca 2+ MPF 42

MII capacitation 20 cumulus cell mass ph 70% 12 ml 3.5 cm 1. 37 30 2. 70% 12 ml BO-washing medium 7 ml BO-washing medium 3000 rpm 10 6 7 ml BO-washing medium 43

BO medium 3. 2. ml BO medium 10 6 /ml 4. 100X BO medium, 10X Chemicals Weight, g Concentration, mm NaCl 6.6553 1138 KCl 0.2997 40.1958 NaH 2 PO 4 H 2 O 0.1145 8.2971 Glucose 2.5181 138.6543 Penicillin 0.6 - Streptomycin 0.68 - Phenol red 0.2% 4 ml 4% MgCl 2 6H 2 O 0.1057 5.1991 CaCl 2 2H 2 O 0.3308 22.4912 Milli Q water Added to 100 ml 100 ml 5. BO-fertilization medium 2 50 µl/droplet COCs BO-fertilization medium 20 30 COCs 50 µl 50 µl 5% CO 2 39 44

6. 6 8 BSA KSOM potassium simplex optimized medium M-199 b synthetic oviductal fluid 12.4 8.12 4.53 mm pyruvatelactate glutamine taurine 20 transgenic animal 45

a b (a) (b) 24 hr (polar body) pb S a b 24 Metaphase II (a) acetic orcein spindle ( ) (b) Telophase II ( ) Pb: S: 46

Biggers, J. D., L. K. McGinnis and M. Raffin. 2000. Amino acids and preimplantation development of the mouse in protein-free potassium simplex optimized medium. Biol. Reprod. 63:281-293. Brackett, B. G. and G. Oliphant. 1975. Capacitation of rabbit spermatozoa in vitro. Biol. Reprod. 12:260-274. Choi, T., F. Aoki, M. Mori, M. Yamashita, Y. Nagahama and K. Kohmoto. 1991. Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos. Development 113:789-795. Eppig, J. J. 1991. Maintenance of meiotic arrest and the induction of oocyte maturation in mouse oocyte-granulosa cell complexes developed in vitro from preantral folliceles. Biol. Reprod. 45:824-830. Ju, J. C., J. E. Parks and X. Yang. 1999. Thermotolerance of IVM-derived bovine oocytes and embryos after short-term heat shock. Mol. Reprod. Dev. 53:336-340. Mattioli, M., G. Galeati, M. L. Bacci and B. Barboni. 1991. Changes in maturation-promoting activity in the cytoplasm of pig oocytes throughout maturation. Mol. Reprod. Dev. 30:119-125. Miyazaki, S. 1988. Inositol 1,4,5-trisphosphate -induced calcium release and guanine nucleotide -binding protein-mediated periodic calcium rises in golden hamster eggs. J. Cell Biol. 106:345-354. Perry, A. C. F., T. Wakayama, I. M. Cooke and R. Yanagimachi. 2000. Mammalian oocyte activation by the synergistic action of discrete sperm head components: induction of calcium transients and involvement of proteolysis. Dev. Biol. 217:386-393. Stice, S. L. and J. M. Robl. 1990. Activation of mammalian oocytes by a factor obtained from rabbit sperm. Mol. Reprod. Dev. 25:272-280. Watanabe, N., G. F. Vande Woude, Y. Ikawa and N. Sagata. 1989. Specific proteolysis of the c-mos proto-oncogene product by calpain on fertilization of Xenopus egg. Nature 342:505-511. 47

1980 DNA DNA gene therapy 48

10 kb DNA position effect DNA DNA DNA BglII KpnI 20 Seo recombinant DNA SV40 DNA SV40 DNA 49

intra-nuclear microinjection Gorden 1980 fibroblast liposome-mediated transfection elcctroporation DNA calcium phosphate copreciptation DEDA- DEDA-dextran carrier protein DNA DNA insert DNA DNA DNA over-expression 50

gene knockout gene replacement homologous sequences gene targeting DNA homologous recombination DNA fibroblast somatic cells primary culture karyotype 1997 Wilmut nuclear transplantation cloned animals stem cells renewal differentiation 51

inner cell mass, ICM embryonic stem cells, ES cells primitive genital ridge primordial germ cells, PGC embryonic germ cells, EG cells adult stem cells severe combined immunodeficiency, SCID plasticity blastocyst injection tissue engineering therapeutic transplantation gene targeting 1981 Evans Kaufman Martin pluripotency 52

proliferation aggregation lineages chimera germline 1998 Thomson 20 HPRT hypoxanthine phosphoribsyl trnasferase CFTR cystic fibrosis transmembrane conductance regulator 53

1966 Pain polymerase chain reaction, PCR Southern blot hybridization 54

gain of function loss of function 55

Chen, L. R., Y. L. Shiue, L. Bertolini, J. F. Merdrano, R. H. BonDurant and G. B. Anderson. 1999. Establishment of plurpotent cell lines from porcine preimplantation embryos. Theriogenology 52:195-212. Chen, L. R., Y. L. Shiue and G. B. Anderson. 1999. Embryonic cell lines derived from morula to late hatched blastocyst stages in pigs. Taiwan Livestock Res. 32:169-181. Evans, M. J. and M. H. Kaufman. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154-156. Martin, G. R. 1981. Isolation of pluripotential cell line from early mouse embryos cultured in medium conditioned with teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 72:1441-1445. 56

Odorico, J. S., D. S. Kaufman and J. A. Thomson. 2001. Multilineage differentation from human embryonic stem cell lines. Stem Cells 19:193-204. Smith, A. G. 2001. Embryo-derived stem cells of mice and men. Annu. Rev. Cell Dev. Biol. 17:435-462. Snouwaert, J. N., K. K. Grigmam, A. M. Latour, N. N. Malouf, R. C. Boucher, O. Smithies and B. H. Koller. 1992. An animal model for cystic fibrosis made by gene targeting. Science 257:1083-1088. Thomson, J. A., J. Itskovitz-E;dor, S. S. Shapiro, M. A. Waknitz, J. J. Swiergiel, V. S. Marshall and J. M. Jones. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282:1145-1147. Vazquez, J. C., C. Nogues, E. Rucker and J. A. Piedrahita. 1998. Factors affecting the efficiency of introducing small mutation in specific genes by homologous recombination; Cre-loxP versus the tag-and-exchange. Transgenic Research 7:181-193. Wall, R. J. 2002. New gene transfer methods. Theriogenology 57:189-201. Wilmut, I., A. E. Schnieke, J. McWhir, A. J. Kind and K. H. Campbell. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385:810-813. Wobus, A. M. 2001. Potential of embryonic stem cells. Mol. Aspects Med. 22:149-164. 57

retrovirus 1970 1980 10% replication-defective adenovirus Adeno-associated virus human immunodeficiency virus, HIV RNA enzyme protein RNA core protein or capsid lipid bilayer protease reverse transcriptase integrase, Int endocytosis RNA cdna integration A B C D C 58

cis- trans- acting sequences, CAS TASCAS long terminal repeat sequences, LTR - promoter enhancer - RNA splicing donor and acceptor psi - RNA TAS gag-pol-env DNA packaging cells help cells gag-pol-env RNA gag-pol-env CMV/GP CMV/VSV-G VSV-G vesicular stomatitis virus glycoprotein VSV-G phospholipid MLV murine leukemis virus ecotropic amphotropic xenotropic MLV MLV HIV VSV-G 1975 59

mosaic 4-8- 2-4- 2- subzona perivitelline space, SPS VSV-G 100% RNA cdna LTR DNA provirus DNA DNA PSNam DNA pgh SVD swine vesicle disease virus, 60

Neomycin pgh EcoRI/BamHI LTR SVD/NeoDNA BamHI DNA DNA gag-pol-env CMV/GP CMV/VSV-G CMV/GP CMV/VSV-G DNA 293T Viagen Inc. CA, USA gag-pol PSNam 293T G418 Neomycin CMV/VSV-G DNA 293T CMV/VSV-G DNA LTR LTR gag-pol-env 10 kb 100 kda SVD DNA SVD DNA RNA internal ribosome entry site, IRES IRES SVD mrna mrna SVD 61

inner cell mass, ICM mosaic germline transmission rate 10% 1-2- 20 200 pl 10 8 cfu/ml 2 20 2- -80 1999 PSNam 10% FCS DMEM DMEM-10 70% 2 3 0.45 µm 25,500 g 4 20 5.5 1/100 DMEM-10 0.2 µm -80 PSNam DMEM-10 90 95% DMEM-10 8 10 0.45 µm 25,500 g 4 5.5 1/100 DMEM-10 0.2 µm -80 1997 Chan DNA 293T 1.5 10 6 T-75 CMV/VSV-G 6 1 2 3 0.45 µm 50,000 g 4 90 50 µl Hank s -80 62

ph VSV-G ph 6.5 6.0 7.2 37 39 39 10% ph 7.4 37 ph 7.4 39 DNA 1-2- ICR ICR 2-cell block polybrene PB PB 8 µg/ml PB PB 8 µg/ml 2-4 µg/ml PB 4 8 µg/ml 10 8 cfu 70% 2 3 90 95% 8 10 ml 10 5 10 6 24 200 63

pl 5 10 10 5 cfu/ml 25,500 g 4 5.5 DMEM-10 M16 DMEM-10 5 10 5 10 5 cfu/ml 100 1/2 200 pl 5 SV-G 10 8 cfu ecotropic amphotropic xenotropic MLV MoMLV Moloney murine leukemis virus HIV-1 Lentivirus Lentivirus SPS 1-75% HIV 10% 1-2- F0 7.4% 2.9% 2- VSV-G F0 82% Lentivirus 1- F0 59.1% LTR Methylation 64

SVD DNA RNA IRES LTR 100 bp 13 pgc histon H1 H3 deacetylation H4 acetylation chromatin tandem repeat DNA rearrangement DNase DNA genetic modification organism, GMO 65

A. MLV B. LTR η Gag/pol Env LTR Receptor-Binding SU Reverse Transcriptase RT Transmembrane TM Intergrase IN trna Lipid bilayer (From Host Cells) Protease PR Matrix MA RNA Genome Capsid CA Nucleoprotein NC (A) (B) C 66

MLV DNA SD=splicing donor, SA=splicing aceptor, G.O.I.= DNA PSNam DNA pgh cdna LTR SVD/Neo EcoRI/BamHI pgh LTR BamHI SVD/Neo 67

( ) 68

1994 1999 28:379-392 Andreadis, S. and B. O. Palsson. 1997. Coupled effects of polybrene and calf serum on the efficiency of retrovial transduction and stability of retroviral vectors. Human Gene Therapy 8:285-291. Chan, A. W. S., E. J. Homan, L. U. Ballou, J. C. Burns and R. D. Bremel. 1998. Transgenic cattle produced by reverse-transcribed gene transfer in oocytes. Proc. Natl. Acad. Sci. USA 95:14028-14033. Chan, A. W. S., K. Y. Chong, C. Martinovich, C. Simerly and G. Schatten. 2001. Transgenic monkeys produced by retroviral gene transfer into mature oocytes. Science 291:309-312. Jaenisch, R. 1976. Germ line integration and Mendelian transmission of the exogenous moloney leukemia virus. Proc. Natl. Acad. Sci. USA 73:1260-1264. Jaenisch, R., H. Fan and B. Croker. 1975. Infection of preimplantation mouse embryos and newborn mice with leukemia virus: tissue distribution of viral DNA and RNA leukemogenesis in the adult animals. Proc. Natl. Acad. Sci. USA 72:4008-4012. Lois, C., E. J. Hong, S. Pease, E. J. Brown and D. Baltimore. 2002. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295:868-872. Miller, O. G., M. A. Adam and A. D. Miller. 1990. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol. Cell Biol. 10:4239-4242. Mizuarai, S., K. Ono, K. Yamaguchi, K. Nishijima, M. Kamihira and S. Iijima. 2001. Production of transgenic quails with high frequency of germ-line transmission using VSV-G pseudotyped retroviral vector. Biochem. Biophys. Res. Commun. 286:456-463. Peters, R. M., B. H. Johnson and R. M. Shuman. 1989. Gene transfer in swine embryos using an avian retrovirus. J. Cell. Biochem., Suppl. 13B:182, Abstract. 69

Peters, R. M., R. M. Shuman, B. H. Johnson and R. V. Mettus. 1987. Gene transfer in swine embryos by injection of cells infected with retrovirus vectors. J. Exp. Zool. 242:85-88. Roe, T., T. C. Reynolds, G. Yu and P. O. Brown. 1993. Integration of murine leukemia virus DNA depends on mitosis. EMBO J. 12:2099-2108. Rubenstein, J. L. R., J. F. Nicolas and F. Jacob. 1986. Introduction of genes into preimplantation mouse embryos by use of a defective recombinant retrovirus. Proc. Natl. Acad. Sci. USA 83:366-368. Schlegel, R. and M. Wade. 1984. A synthetic peptide corresponding to the NH 2 terminus of vescular stomatitis virus glycoprotein is a ph-dependent hemolysin. J. Biol. Chem. 259:4691-4694. 70

RNAi RCAS-TVA 1 2 1 2 RNA RNA interference, RNAi RNA mrna RNA Dicer 21 25 酶 RNA small interfering RNA, sirnasirna mrna RdRP sirna gene silencing effect RNAi RNAi bioreactor RNAi RNA RCAS -TVA RCAS-TVA NIH Harold Varmus ALV avian leukosis virus TVA ALV RCAS TVA RCAS-TVA RNAi gain-of-function loss-of-function 71

gene silence gene knock out DNA posttranscription mrna RNA antisense ribozyme gene knock down RNAi RNA interference RNA sense RNA target gene RNAi RCAS-TVA Rous sarcoma virus src src gene of interest TVA RNAi RCAS-TVA RNAi RNAi RNAi 1996 Rich Jorgensen chalcone synthase petunia endogene posttranscriptional gene silence, PTGS par-1 mrna C. elegans RNA 72

par-1 mrna par-1 mrna mrna mrna RNA homology RNAi RNA RNAi hairpin RNA RNAi RNAi RNAi RNAi RNA RNA dsrna-activated protein kinase PKR PKR translation initiation factor mrna RNA 2 5 -AS/RnaseL mrna apoptosis RNAi mrna 21 25 sirna, small interfering RNA sirna RNA RNA RNAi RNA RNAi sirna DNA 73

RNA insert DNA 25 30 RNA RNA small hairpin RNA shrna sirna DNA inverted repeats RNA 19 9 ( ) U6 U6 polymerase promoter H1 Histone1-promoter sirna termination signal TTTTT sirna http://www.ambion.com/techlib/tn/94/9420.html?srcd=aaa S0209 RNAi RNAi RNA RNA mrna RNAi RNA RNAi RNAi RNA RNAi RNA RNAi RNAi transposon RNAi RNAi (C. elegans) RNA 74

Initiation step RNA trigger Dicer 21 25 nt guide sequence sirna Dicer RNaseIII RNA dsrna-specific endonuclease Effector step sirna nuclease complex RNA- induced silencing complex/risc sirna mrna RDE-4 RNA RNA RNA Dicer sirna RNA RNA RNA Dicer sirna 3 sirna RNA RNAi RNA mrna RNA Nishikura Lipardi Nykanen RNAi DNA PCR replication (oligonucleotide) RNA RNAi Dicer 21 25 mer sirna sirna mrna mrna RNA RNA dependent RNA polymerase, RdRP RNA 75

RNA Dicer 21 25 mer sirna mrna RNAi RNA Dicer sirna RNAi RdRP sirna sirna mrna RNAi primary RNAi transitive RNAi silence RNAi RdRP RNA RdRP RNA RNAi RNAi gene knock out - DNA ES multipotent ES 76

PCR RNAi mrna RNAi RNA RNAi RNAi ES RNAi 1. RNAi sirna mrna mrna mrna alternative splicing mrna RNAi RNA transitive RNA RNAi (cross RNAi) B GFP GFP-lacZ RNA lacz RNAi GFP A GFP lacz RNA RNAi redundancy compensation RNAi RNAi mrna 77

shrna RNA RNAi 2. RNAi RNA, RNAi 3. RNAi Lee 2002 RNAi HIV-1 rev HIV-1 RNA RNAi RNAi crown gall disease Sindbis virus dengue virus Aedes aegypti RNAi RNA RNA sirna RNAi RNA RNAi sirna DNA shrna C 78

sirna shrna RNAi 4. bioreactor RNA RCAS-TVA A avian leukosis virus subgroup A/ALV-A RNA RNA DNA DNA RNA DNA HIV genomic structure DNA DNA genomic DNA DNA ALSV, avian leukosis and sarcoma virus family RSV ALV src LTR U3 R LTR R U5 RNA RNA genomic RNA RNA Gag Gag-Pol polyproteins mrna gag splice donor site env splice acceptor site splicing RNA Env mrna RSV src mrna 79

Src TVA TVA tv-a ALSV, avian leukosis and sarcoma virus family LDLR low-density lipoprotein receptor TVA ALSV TVA TVA ALSV TVA ALSV ALSV ALV-A TVA α-actin-tv-a β-actin-tv-a GFAP-tv-a astrocytes nestin-tv-a glial progenitors GP-Iba-tv-a megakaryocyte lineage DCT-tv-a melanoblasts RCAS ALSV RSV Schmidt-Ruppin A RSV RSV mrna Src src ClaI X 2.5 80

kb 3.0 kb RCAS DF1 Doug Foster CEF LTR protein of interest RNA virion TVA LTR virion RCAS LTR LTR 293 LTR gene of interest RCAN RCAN RCAS LTR LTR TVA ALV-A ALV-A env env ALV-A ALV-A ALV-A 81

TVA RCAS-TVA 1. RACS-TVA TVA 2. RACS-TVA gene of interest 3. TVA 4. RCAS-cDNA (http://rex.nci.nih.gov/research/basic/varmus/tva-we b/tva2.html) 5. RCAS 6. TVA 82

RCAS-TVA ALV-TVA ALV-A ALV-TVA ALV lentivirus genomic structure TVA HIV ALSV lentivirus HIV HIV genome nuclear localization signal NLS DNA RCAS-TVA RCAS-TVA RNAi RCAS-TVA RCAS X shdna shrna DNA shdna RNAi shrna RCAS RCAS-TVA TVA 83

RCAS-TVA RNAi RCAS-TVA RNAi RNAi sirna RCAS-TVA shrna Nishikura HIV Drug Resistance Program, National Cancer Institute Nature Publishing Group Hughes RCAS-TVA 84

RNAi T5 TTTTT ( ) H1 promoter U6 promoter shrna RNAi RNA mrna - ; - ; - ; -RDE-4 1st sirna 1st sirna primary sirna secondary sirna RNAi A sirna B sirna RNA 85

"Reprinted from http://home.ncifcrf. gov/hivdrp/rcas/http://home.ncifcrf.gov/ hivdrp/rcas/ by permission of the HIV Drug Resistance Program, National Cancer Institute" "Reprinted from http://home. ncifcrf.gov/hivdrp/rcas/ by permission of the HIV Drug Resistance Program, National Cancer Institute" 86

RCAS ( gene X ) ClaI "Reprinted from http://home.ncifcrf.gov/hivdrp/rcas/ by permission of the HIV Drug Resistance Program, National Cancer Institute" A B A DF-1 TVA RCAS DF-1 DF-1 TVA "Reprinted from http://home.ncifcrf.gov/ hivdrp/rcas/ by permission of the HIV Drug Resistance Program, National Cancer Institute" 87

Adelman, Z. N., C. D. Blair, J. O. Carlson, B. J. Beaty and K. E. Olson. 2001. Sindbis virus-induced silencing of dengue viruses in mosquitoes. Insect Mol. Biol. 10:265-273. Fisher, G. H., S. ORsulic, E. Holland, W. P. Hively, Y. Li, B. C. Lewis, B. O. Williams and H. E.Varmus. 1999. Development of a flexivle and specific gene delivery system for production of murine tumor models. Oncogene 18:5253-5260. Guo, W., S. C. Winistorfer and C. M. Stoltzfus. 2000. Selective inhibition of splicing at avian sarcoma virus src 3 splice site by direct-repeat posttranscriptional cis elements. J. Virol. 74:8513-8523. Hannon, G. J. 2002. RNA interference. Nature 418:244-251. Jacque, J. M., K. Triques and M. Stevenson. 2002. Modulation of HIV-1 replication by RNA interference. Nature 418:435-438. Lee, N. S., T. Dohjima, G. Bauer, H. Li, M. J. Li, A. Ehsani, P. Salvaterra and J. Rossi. 2002. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat. Biotechnol. 20:500-505. Lewis, B. C., N. Chinnasamy, R. A. Morgan and H. E. Varmus. 2001. Development of an avian leucosis-sarcoma virus subgroup A pseudotyped lentiviral vector. J. Virol. 75:9339-9344. McCaffrey, A. P., L. Meuse, T. T. Pham, D. S. Conklin, G. J. Hannon and M. A. Kay. 2002. Gene expression: RNA interference in adult mice. Nature 418:38-39. Smith, N. A., S. P. Singh, M. B. Wang, P. A. Stoutjesdijk, A. G. Green and P. M. Waterhouse. 2000. Total silencing by intron-spliced hairpin RNAs. Nature 407:319-320. 88

transgenic animal DNA SV40 DNA 78 2 Embryonic stem cells, ES cells SV40 SV40 DNA DNA 30 30% DNA 21% DNA 89

DNA DNA DNA autoradiography SV40 DNA DNA SV40 SV40 DNA Abalone Lucilia cuprina Apis mellifera Xenopus laevis Carp Catfish Salmon Loach Tilapia Zebra fish DNA DNA DNA 9 / 4500 / DNA 30 DNA 32 P DNA 5% DNA DNA post-acrosomal region DNA DNA DNA DNA 41 80% DNA 395 845 DNA DNase DNA DNA 24 56 DNA 32 P DNA 39% 355/913 DNA equatorial segment 1% DNA 90

DNA DNA DNA DNA 30 15 20% DNA DNA 30 35 kd DNA DNA DNA rearrangement DNA II major histocompatibility complex II CD4 DNA 酶 DNA DNA DNA apoptosis DNA fragmentation DNA DNA DNA DNA 30% DNA zona pellucida, ZP perivitelline space microvilli 91

male pronucleus DNA DNA DNA DNA DNase DNA 56 DNA DNA 2000 volts/0.4 cm electroporation DNA 98 Southern blot hybridization DNA DNA 186 DNA DNA DNA PCR polymerase chain reaction, PCR DNA DNA liposome DNA 11 13 25.9% DNA PCR DNA 92

PCR DNA DNA DNA DNA episome DNA 30% 酶 酶 酶 酶 酶 DNA 酶 酶 DNA DNase DNA DNA DNA DNA nucleus-like 93

DNA DNA DNA DNA DNA DNA DNA DNA 1. DNA DNA DNA/liposome complex DNA 2. DNA DNA 3. DNA DNA linker protein DNA 33 37.5% 94

4. DNA DNA DNA DNA testis-mediated gene transfer 1. DNA 2. DNA DNA DNA gamate intrafallopin transfer 3. in vitro fertilization, IVF 4. intracytoplasmic sperm injection DNA DNA chromatin mosaicism DNA DNA 95

96

DNA DNA Southern 97

1996 24:427-440 1995 DNA 24:289-300 Atkinson, P. W., E. R. Hines, S. Beaton, K. I. Matthaei, K. C. Reed and M. P. Bradley. 1991. Association of exogenous DNA with cattle and insect spermatozoa in vitro. Mol. Reprod. Dev. 29:1-5. Brinster, R. L., E. P. Sandgren, R. R. Behringer and R. D. Palmiter. 1989. No simple solution for making transgenic mice. Cell 59:239-241. Chang, K., J. Qian, M. Jiang, Y. H. Liu, M. C. Wu, C. D. Chen, C. K. Lai, H. L. Lo, C. T. Hsiao, L. Brown, J. Jr. Bolen, H. I. Huang, P. Y. Ho, P. Y. Shih, C. W. Yao, W. J. Lin, C. H. Chen, F. Y. Wu, Y. J. Lin, J. Xu and K. Wang. 2002. Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer. BMC Biotechnol. 2:5. Gordon, J. W., G. A. Scangos, D. J. Plotkin, J. A. Barbosa and F. H. Ruddle. 1980. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. USA 77:7380-7384. Hammer, R. E., V. G. Pursel, C. E. Rexroad Jr., R. J. Wall, D. J. Bolt, K. M. Ebert, R. D. Palmiter and R. L. Brinster. 1985. Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315:680-683. Lavitrano, M., A. Camaioni, V. M. Fazio, S. Dolci, M. G. Farace and C. Spadafora. 1989. Sperm cells as vectors for introducing foreign DNA into eggs: Genetic transformation of mice. Cell 57:717-723. Lavitrano, M., D. French, E. Forte, M. Francolini, S. Sperandio, R. Testi and C. Spadafora. 1997. The interaction of sperm cells with exogenous DNA: a role of CD4 and major histocompatibility complex class II molecules. Exp. Cell Res. 233:56-62. Zani, M., M. Lavitrano, D. French, V. Lulli, B. Maione, S. Sperandio and C. Spadafora. 1995. The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake. Exp. Cell Res. 217:57-64. 98

1 2 1 2 human decay accelerating factor, hdaf 99

α- α-1,3-galactosyltransferase α-gt human factor IX, hfix1996 hfix factor IX complex hfix 1994 28 (transmissible spongiform encephalopathy) Prion-free 1966 100

1980 HSV herpes simplex virus SV40 simian virus 40 DNA 5 15% DNA rat mouse mmt-hgh DNA Lavitrano 1989 20 30% 50 60% DNA DNA 101

reprogram 1997 Wilmut Dolly PPL hfix Polly 2002 hfix 0.2 2% 4-8- 16 24 1-10 kb embryonic stem cells, ES cells inner cell mass, ICM 102

ESC gene targeting gene knockout DNA DNA DNA SDS NaOH CsCl banding DNA RNA 103

DNA DNA DNA Banding DNA TE 10 mm Tris/0.1 mm EDTA, ph=7.4 8.0 1 5 ng/µl GMO DNA DNA 0.22 µm 20 30 µl -80 1. PGF2α 11 12 13 14 13 14 PGF2α 2. regumate 0.4% Altrenogest 20 mg 18 15 1 3 PGF2α 3. 11 12 11 15 PMSG HCG PMSG 24 HCG PMSG HCG 72 80 104

72 80 76 78 HCG HCG 52 58 4 6 9 11 7 8 HCG 6:30 12 3 HCG 8:00 12 3 21 10 4 6 9 PMSG/HCG Intervet 1500/1250 IU2000/1750 IU 500/500 IU 5 6 PMSG/HCG PMSG/HCG 7 8 FDA 105

HCG 54 56 HCG 1. 18 20 1 cm 25 ml D-PBS 0.1% BSA / 2. 5 cm 1 cm 18 25 ml D-PBS 0.1% BSA / 25 30 10 ml 106

15,000 23,500 g 8 10 150 µm 75 10 mm DNA 0.6 µl DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA HCG 54 56 HCG 60 107

DNA DNA DNA HCG 54 56 germinal vesicle, GV PMSG HCG GV PMSG/HCG 1 2 PGF2α Kliev s 30 37 108

18 20 24 15 2 DNA DNA PCR PCR PCR nitrocellulose membrane DNA NC 32 P-dCTP gene 109

integration DNA 酶 rearrangement mrna Northern blot mrna RT-PCR RT-PCR Northern blot RT-PCR ELISA Western blot hybridization imunohistochemical staining, IHC Flowcytometric analysis, FACSELISA IHC FACS APTT activated partial thromboplastin time Beagle 110

DNA HPLC mrna hemizygote 50% DNA F1 F2 PMSG/HCG 3 4 111

CMV β-actin Metallothionein MT β-actin CMV MT β- β-casein why acidic protein α- α-alactoalbunimβ- β-lactoglobulin β- 1 mg/ml CMV β-actin Enviropig TM rearrangement 112

113

0 1-15 16 10:00 20 6:30 21 10 4 22 1:30 134 (S) (R) 2000IU PMSG (P) 1500IU hcg (H) (A.I) (M) SPF (F) D0 D1 D15 D16 D20 D21 S R R P H A.I. M F 120 kg ( ) 25 µm 114

Brem, G., B. Brenig, H. M. Goodman, R. C. Selden, F. Graf, B. Kuff, K. Springman, J. Hondele, J. Meyer, E. L. Winnacker and H. KarauBlich. 1985. Production of transgenic mice, rabbits and pigs by microinjection foreign DNA into pronuclei. Zuchthygiene 20:251-252. Brinster, R. L., H. Y. Chen, M. E. Trumbauer, M. K. Yagle and R. D. Palmiter. 1985. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. USA 82:4438-4442. Cabot, R. A., B. Kuhholzer, A. W. S. Chan, L. Lai, K. W. Park, K. Y. Chong, G. Schatten, C. N. Murphy, L. R. Abeydeera, B. N. Day and R. S. Prather. 2001. Transgenic pigs producing in vitro matured oocytes infected with a retroviral vector. Anim. Biotech. 12:2005-2014. Gorden, J. W., G. A. Scangos, D. J. Plotkin, J. A. Barrosa and F. H. Ruddle. 1980. Genetic transformation of mouse embryos by microinjection od purified DNA. Proc. Natl. Acad. Sci. USA 77:7380-7384. Hammer, R. E., V. G. Pursel, C. E. Rexoad, Jr., R. J. Wall, D. J. Bolt, K. M. Ebert, R. D. Palmiter and R. L. Brinster. 1985. Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315:680-683. Kupriyanov, S., K. Zeh and H. Baribault. 1998. Double pronuclei injection of DNA into zygotes increases yields of transgenic mouse lines. Transgenic Res. 7:223-226. Lavitrano, M., A. Camaioni, V. M. Fazio, S. Dolci, M. G. Frace and C. Spadafora. 1989. Sperm cells as vector for introducing foreign DNA into eggs: genetic transformation of mice. Cell 57:712-723. Lavitrano, M., M. Forni, V. Varzi, L. Pucci, M. L. Bacci, C. D. Stefano, D. Fioretti, G. Zoraqi, B. Moioli, M. Rossi, D. Lazzereschi, A. Stoppacciaro, E. Seren, D. Alfani, R. Cortesini and L. Frati. 1997. Sperm-mediated gene transfer: production of pigs transgenic for a human regulator of complement activation. Transplant Proc. 29:3508-3509. Lin, T. P. 1996. Microinjection of mouse embryo. Science 151:333-337. Martin, M. J. and C. A. Pinkert. 1993. Production of transgenic swine. In: Transgenic Animal Technology: A Laboratory Handbook, pp. 315-338. Academic Press, Inc., USA. Palmiter, R. D., R. L. Brinster, R. E. Hammer, M. E. Trumbauer, M. G. Rosenfeld, N. C. Brinberg and R. M. Evans. 1982. Dramatic 115

growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300:611-615. Wilmut, I., A. E. Schnieke, J. Mcwhir, A. J. Kind and K. H. S. Campbell. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385:810-813. 116

1 2 1 2 1997 Genzyme Transgenics 60 US$100-1000 US$100 10-25 cents 30,000 60,000 DNA DNA 117

250 3 g 400 mg IgY TranXenoGen AviGenics DNA primordial germ cells, PGC 5 6 22 1976 Eyal-Giladi Kochav I-XIV 14 1951 Hamburger Hamilton 1 46 Eyal-Giladi Kochav X stage X area pellucida area opaca epiblast 118

hypoblast stage XI-XIII stage XIV Hamburger Hamilton stage 2 primitive streak endoderm germinal crescent PGC Stage X PGC X PGC gonadal ridges PGC PGC PAS periodic acid-schiff glycogen PGC EMA-1 SSEA-1 PGC PGC SSEA-1 ex vivo culture 1988 Perry I II III I II III I 41 42 24 24 119

II 24 96 38 90 III 96 38 30 10% III 40% II III 20% Naito I II 30% DNA DNA DNA DNA 15 5 4 8 4 256 120

20 60,000 2.5 25 30 10 15 µm DNA PGC DNA 0.1 mg/ml 3 nl 10 50 nl lacz gene β-actin-lacz 4 63.6% Perry lacz gene 12 ex vivo 25 DNA DNA DNA DNA 10 genome copy G1 3.4% G2 81.5% DNA 0.8% PGC PGC PGC X 121

PGC PGC germline chimera PGC DNA PGC PGC Hamburger Hamilton 13 14 4 5 PGC PGC 14 19 27 28 7 9 PGC PGC PGC PGC DNA transfection DNA PGC DNA PGC PGC PGC PGC PGC DNA PGC 122

PGC 22% 59% 10 95% donor PGC 0.1% 96% PGC Injected embryos Source of PGCs 372 Gonadal PGCs 145 Circular PGCs Hatched chicks (%) No reach sexual maturity Germ line Chimeric chicks (%) Donor-de.%* 219(59) 73 6/60(10) 1.3-3.1% 32(22) 20 19/20 (95) 2-96% Incubation Original egg shell incubation Ex vivo incubation Ref. Chang, et. al., 1997 Naito, et. al., 1994 314 Circular PGCs 83(26) 74 31/74(42) 0.1-40% *donor-de.%: Percentage of chicks with donor-derived plumage Ex vivo incubation Naito, et. al., 1999 blastomeres stage X 2 DPBS hairloop CMF-PBS 10 0.05% trypsin 5 10 DMEM 10% FBS 2 24 500 PGC 5 15% ex vivo 30% 123

I I i i I I i DNA DNA 4 4.9 8.7% 0 63% PGC donor 100% Injected embryo Days of incubation Alive embryos (%) Hatched chicks (%) Somatic chimeric chicks (%) Germ line chimeric chicks (%) Donor-de*.% Treatment Ref. 1017 8 311(30) 50(4.9) 13(26) 3/13(23) 2.9-30.7% Recipient γ irradiation Thoraval et. al., 1994 712 8 253(35) 52(7.3) 23(44) 0 No γ irradiation 298 14 63(21) 26(8.7) 15/23(65) 7/20(35) 1-99% 20/27(74) 17/27(63) 6-100% FACS-sorted & 60Co 500 rads Remove recipient CBCs first Thorava et. al., 1994l Speksnijder et. al.,1999 Kagami et.al., 1997 *donor-de.%: Percentage of chicks with donor-derived plumage PGC fluorescence-activated cell sorting FACS FACS 124

PGC 48 homologous recombination PGC PGC 1989 Lavitrano DNA DNA 75 7% 130/1755 PCR Southern blot hybridization ICSI intracytoplasmic sperm injection DNA GFP DNA REMI restriction enzyme-mediated integration 125

TMGT testis-mediated gene transfer DNA DNA lipofection 126

I 24 II 24-96 III 127

stage X donor () recipient stage X ( ), DNA 128

Chang, I., D. K. Jeong, Y. H. Hong, T. S. Park, Y. K. Moon, T. Ohno and J. Y. Han. 1997. Production of germline chimeric chicken by transfer of cultured chick primordial germ cells. Cell Biol. Int. 21:495-499. Eyal-Giladi, H. and S. Kochav. 1976. From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick. I. General Morphology. Dev. Biol. 49:321-337. Hamburger, V. and H. L. Hamilton. 1951. A series of normal stages in the development of the chick embryo. J. Morph. 88:49-92. Kagami, H., T. Tagami, Y. Matsubara, T. Harrumi, H. Hanada, K. Maruyama, M. Sakurai, T. Kuwana and M. Naito. 1997. The developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mix-sex germline chimeras to the offspring in the chicken. Mol. Reprod. Dev. 48:501-510. Love, J., C. Gribbin, C. Mather and H. Sang. 1994. Transgenic birds by DNA microinjection. Biotechnology 12:60-63. Naito, M., A. Tajima, Y. Yasuda and T. Kuwana. 1994. Production of germline chimeric chickens, with high transmission rate of donor-derived gametes, produced by transfer of primordial germ cells. Mol. Reprod. Dev. 39:153-161. Naito, M., K. Agata, K. Otsuka, K. Kino, M. Ohta, K. Hirose, M. M. Perry and G. Eguchi. 1991. Embryonic expression of β-actin-lacz hybrid gene injected into the fertilized ovum of the domestic fowl. Int. J. Dev. Biol. 35:69-75. Naito, M., Y. Matsubara, T. Harrumi, T. Tagami, H. Kagami, M. Sakurai and T. Kuwana. 1999. Differentiation of donor primordial germ cells into functional gametes in the gonads of mixed-sex germline chimeric chickens produced by transfer of primordial germ cells isolated from embryonic blood. J. Reprod. Fert. 117:291-298. Nakanishio, A. and A. Iritani. 1993. Gene transfer in the chicken by sperm-mediated methods. Mol. Reprod. Dev. 36:258-261. Perry, M. M. 1988. A complete culture system for the chick embryo. Nature 331:70-72. Sang, H. 1994. Transgenic chickens- methods and potential applications. Trends in Biotechnology. 12:415-420. Speksnnijder, G. J., R. J. Etches and A. M. V. Gibbins. 1999. Germline chimeric chickens from FACS-sorted donor cells. Mol. Reprod. Dev. 52:33-42. Thoraval, P., F. Lasserre, F. Coudert and G. Dambrine. 1994. 129

Somatic and germline chicken chimeras obtained from brown and white Leghorns by transfer of early blastodermal cells. Poultry Sci. 73:1897-1905. 130

DNA DNA 40 kit DNA DNA temporal spatial DNA DNA cell lineage reporter gene β-galactosidase gene lacz, 35 kda tetramer horseradish peroxidase HRP human placental alkaline phosphatase hplap, hap in vivo DiI 1,1'-dioctadecyl-3,3,3',3'-tatramethylindocarbo-cyanine colloidal gold LRD lysinated rhodamine dextran DNA fluorescence phosphorescence jellyfish green fluorescent protein, GFP non-invasive real time in situ 10 131

Green Fluorescent Protein Aequorea victoria wild-type, wt T m =70 37 folding excitation wavelength 395 nm emission wavelength 509 nm 470 nm 540 nm high-pressure mercury short arc lamp wt GFP 1996 Cormack Yang mutant, mt enhanced GFP, EGFP Heim and Tsien cyan Ormo 1962 Shimomura 1974 GFP 1978 27 28 kda 1979 chromophore 1996 GFP 3-dimentional structure Prasher 1992 wt GFP 1994 GFP 238 7-229 β-can 24 Å 42 Å 1 Å=10-10 m 64Phe-Ser-Tyr-Gly-Val-Gln69 65Ser-Tyr-Gly67 11 β-sheet α-helix GFP 90 4 EGFP 132

24 wt GFP ph 5.5-12.0 EGFP ph 7.0 11.5 GFP denaturant 8 M urea 1% SDS glutaraldehyde formaldehyde agarose rubber cement GFP wt GFP EGFP GFP monomer dimer EGFP 100 nm 10,000 2,000 GFP Discosoma sp. Heteractis crispa vector fusion protein chimeric protein 37 10-7 10-9 50 100 nm Stokes shift 10-4 10-6 photogenic cell aequorin 酶 luciferase luciferin, coelenterazine 3 Ca 2+ Ca 3 -apo-aequorin-coelenteramide in vitro GFP autofluorescence Gly67 amide group Ser65 carbonyl group imidazoinone 133

H 2 O O 2 Tyr66 α-β bond 4-(ρ-hydroxybenzy lidene)-imdazolid-5-one GFP on/off blinking photongfp dark state 5 GFP photobleachingresistance wt GFP GFP Protein Databank 24 GFP BFP CFP YFP RFP complementary color multicolor fluorescence EGFP+RFP ECFP+EYFP ECFP+EYFP+RFP ECFP EYFP two-colored fluorescent mouse flow cytometry 458 nm ECFP EGFP EYFP 488 nm EGFP EYFP DsReddual-laser 458 nm 586 nm ECFP EGFP EYFP DsRed nm 400-450 450-500 500-550 550-600 600-650 650-750 magenta 134 cyan

R G BB+G C B+R M G+R Y CMY a, / b (nm) b EBFP ECFP wt GFP EGFP EYFP DsRed2 HcRed1 382 excitation 434/453 395/470 489 514 561 588 444 emission 477/501 509/540 508 527 587 618 4 ( ) (absorbance )extinction ~28.6 26 9.5 55 84 43.8 20 coefficient (,10 3 M -1 cm -1 ) quantum yield (QY,, %) 22 40 80 60 61 55 1.5 1. Patterson 2001Clontech 2001 2002 2. absorbance, A A/bc b cm c /L 3. 4. intensity x QY a b wt GFP regulatory sequence GFP GFP GFP 135

turnover rate defp destabilized enhanced fluorescent protein 1 4 fusion tag GFP amino carboxyl host protein DNA localization dynamic fate DNA 1994 GFP E. coli Caenorhabditis elegans Dyctyostelium and Arabidopsis thaliana Drosophilia melanogaster GFP cardiac α-myosin heavy chain gene, α-myhc, promoter GFP GFP dose-dependent wt GFP GFP wt GFP EGFP wt GFP 64 Phe Leu 65 Ser Thr190 silent base codon humanized EGFP 488 nm wt GFP 35 1997 Okabe pcx-egfp microinjecting pronucleus 136

pcx-egfp embryonic stem cells, ES cells aggregation ECFP EYFP 2002 pcx-egfp blastocyst 4n tetraploid embryo detection laser-scanning confocal quantitative fluorometric assay transmitted incident intensity 1. high-pressure mercury short arc lamp 300 600 nm line spectrum 337 365 405 436 546 578 nm 10 nm background emission HBO 50 W/AC arc size 1.0 0.3 mm 200 V 2,000 lumens, lm 30,000 candela/cm 2, cd/cm 2 100 W/AC arc size 0.25 0.25 mm 45 V 2,200 lm 170,000 cd/cm 2 100 200 20 137

2. high-pressure xenon short arc lamp 300-800 nm continuous spectrum XBO 75 W/2 arc size 0.5 0.25 mm DC 45 50 V 1,000 lm 40,000 cd/cm 2 400 1. absorption filter 30 250 nm transmittance glass, G colored filter 520 nm Zeiss G 436 405 490 nm 2. band-pass BP interference filter 10 nm 138

BP 450-490 450 490 nm BP 485/20 475 495 nm 90% excitation filter 3. short-pass SP SP 490 490 nm 4. long-pass LP LP 520 520 nm barrier filter 5. chromatic beam splitter, CBS dichromatic mirror, DM optical axis 45 90%CBS 510 510 nm 510 nm condenser fluorite semi-apochromats numerical aperture NA NA flavin FAD FMN 450 515 nm GFP PBS 139

1. Zeiss Axiovert 35 EGFP UVG-50 OSRAM HBO 50 W/AC, 200 V filter set 09, Cat. No. 4879094 BP 450-490 exciter filter 450 490 nm dichromatic beam splitter FT 510 EGFP 488 490 nm 507 509 nm 510 nm barrier filter LP 520 nm 520 530 nm 2. Kodak Ektachrome P1600 color reversal film = ASA 400 800 Contax 167/MT UVP 365 nm Nikon F-401 Nikon AF MICRO NIKKOR 55 mm, 1:2.8 520 nm 140

green fluorescent protein, GFP 10 non-invasive real time in situ wt GFP 37 wt GFP 1996 EGFP fusion tag 1997 2002 4n tetraploid embryo 2002 ECFP EYFP ECFP EGFP EYFP RFP laser-scanning confocal 141

300-400 400-450 450-500 500-550 550-600 600-650 650-750 750-900 ( ) (nm) 142

Bevis, B. J. and B. S. Glick. 2002. Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat. Biotechnol. 20:83-87. Chalfie, M. and S. Kain. 1998. Green Fluorescent Protein: Properties, Applications, and Protocols. Wiley-Liss, NY, USA. Clontech Lab., Inc. 2001, 2002. Living Colors, User Manual PT2040-1, PT3404-1. http://www.clontech. com/techinfo/manuals/index.shtml Hadjantonakis, A. K. and A. Nagy. 2001. The color of mice: In the light of GFP-variant reporters. Histochem. Cell Biol. 115:49-58. Hadjantonakis, A. K., S. Macmaster and A. Nagy. 2002. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal. BMC Biotechnol. 2:11-19. Hawley, T. S., W. G. Telford, A. Ramezani and R. G. Hawley. 2001. Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins. Biotechniques 30:1028-1034. Huang, W. Y., J. Aramburu, P. S. Douglas and S. Izumo. 2000. Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy. Nat. Med. 6:482-483. Matz, M. V., A. F. Fradkov, Y. A. Labas, A. P. Savitsky, A. G. Zaraisky, M. L. Markelov and S. A. Lukyanov. 1999. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17:969-973. Patterson, G., R. N. Day and D. Piston. 2001. Fluorescent protein spectra. J. Cell Sci. 114:837-838. Zimmer, M. 2002. Green fluorescent protein (GFP): Application, structure, and related photophysical behavior. Chem. Rev. 102:759-781. 143

gene transfer terminator transgenic fish medaka, Oryzias latipes zebrafish, Danio rerio 2 3 20% polyethylene glycol liposome embryonic-derivated stem cells microinjection electroporation particle bombardment sperm-mediated gene transfer 144

proteinase trypsin micropyl DNA puller grider 10 µm DNA injection manupilator 5 10 pl 10 holding manupilator DNA DNA DNA DNA 1/10 DNA DNA RSV-LTR galactosidase lacz 145

12 40% ( ) Acanthopagrus schlegeli Penaeus monodon Macrobrachium rosenbergii GH GH GH copies GH GH cdna GH cdna 37.0 10.2 g 5.94 14 g6 76.7 g 13 50%14 161 DNA2.5 146

DNA 0 52% 26% 3% 15 33% Southern blot hybridization DNA mosaicism 147

3 5 cm, 148

WTO American Fisheries Society DNA 149

1995 Frankenfish Salmo salar Aqua Bounty Farms 1999 coho salmon Oncorhynchus kisutch coho salmon 1999 1 1000 40 150

in vivo 151

6 ( ) 2.5 ( ) ( ) 6 ( ) DNA 152

DNA ( ) DNA DNA 153

Hsiao, C. D., F. J. Hsieh and H. J. Tsai. 2001. Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish. Dev. Dynam. 220:323-336. Li, S. S. and H. J. Tsai. 2000 Transfer of foreign gene to giant freshwater prawn Macrobrachium rosenbergii by spermatophore- microinjection. Mol. Reprod. Dev. 56:149-154. Lu, J. K., T. T. Chen, C. L. Chrisman, O. M. Andrisani and J. E. Dixon. 1992. Integration, expression, and germ-line transmission of foreign growth hormone genes in medaka Oryzias latipes. Mol. Marine Biol. Biotechnol. 1:366-375. Ma, G. C., T. M. Wang, C. Y. Su, Y. L. Wang, S. Chen and H. J. Tsai. 2001. Retina-specific cis-elements and their binding nuclear proteins of carp rhodopsin gene. FEBS Lett. 508:265-271. Tsai, H. J., C. H. Lai and H. S. Yang. 1997. Sperm as a carrier to introduce an exogenous DNA fragment into the oocyte of Japanese abalone Haliotis disvorsicolor suportexta. Transgenic Res. 6:85-95. Tsai, H. J. and F. S. Tseng. 1994. Electroporation of a foreign gene into black porgy Acanthopagrus schlegeli embryos. Fish. Sci. 60:787-789. Tsai, H. J., F. S. Tseng and I. C. Liao. 1995a. Electroporation of sperm to introduce foreign DNA into the genome of loach Misgurnus anguillicaudatus. Can. J. Fish. Aquat. Sci. 52:776-787. Tsai, H. J., S. H. Wang, K. Inoue, S. Tskagi, M. Kimura, Y. Wakamatsu and K. Ozato. 1995b. Intiation of the transgenic lacz gene expression in medaka Oryzias latipes embryos. Mol. Marine Biol. Biotechnol. 4:1-9. Tseng, F. S., H. J. Tsai, I. C. Liao and Y. L. Song. 2000. Introducing foreign DNA into tiger shrimp Penaeus monodon by electroporation. Theriogenology 54:1421-1432. Tseng, F. S., I. C. Liao and H. J. Tsai. 1995. Transient expression of mouse tyrosinase gene in catfish albino mutants Clarias fuscus by subcutaneous microinjection. Fish. Sci. 61:163. 154

1 2 1 2 gene knockout gene targeting homologous recombination DNA DNA target gene 1985 β-globin 1981 Kaufmann Martin mouse embryonic stem cell line blastocyst foster mother 1987 Smithies Capecchi HPRT int-2 c-abl A targeting vector electroporation antibiotics heterozygous, +/- microinjection B 155

A B coat color chimeric mice F1 wild-type, wt F2 +/- germ line chimera A +/- 1/4 homozygous, -/- F3 -/- phenotype inner cell mass, ICM pluripotency uncommitted germ line transmission feeder cells leukaemia inhibitory factor, LIF genetic background 129 129 156

C57BL/6 BALB/C rat targeting vector isogenic genomic DNA library λ λ phage, 15 20 kb cosmid 35 50 kb 1. A gene cassette reading frame antibiotic-resistant gene neomycin puromycin hygromycin promoter PGK CMV poly A adenylation signal DNA SV40-poly A BGH-polyA A DNA restriction enzyme linearlization DNA 157

transcriptiontranslation incomplete knockout replacement 2. replacement B functional domain open reading frame DNA B DNA long arm short arm positive selection marker negative selection marker thymidine kinase, TK diphtheria, DT-A gancyclovir FIAU 1-(2-deoxy- 2-fluoro-β-Darabino-furanosyl)-5-iodouracil TK gancyclovir FIAU analog DT-A DT-A 3 10 backbone multiple cloning sites, MCS 158

reporter gene β-galactosidase lacz electroporation ori kb 15 20 kb 96-well 24-well DNA DNA Southern blot hybridization PCR probe PCR primer PCR noninvasive 129/D3 agruti C57BL/6 129 C57BL/6 C57BL/6 +/- 159

+/- dosage effect -/- / DNA 1. incomplete knockout 5 - N- N- associate proteins C- signal transduction transdominate effect 1 160

cryptic 2 AUG AUG N- 3 exon splicing phenotype 2. coding region DNA intron complementary strain 3. DNA Cre FLP recombinase 4. 1 161

multiple gene knockout 2 129 129 C57BL/6 129 BALB/C 129 129 129 C57BL/6 BALB/C folding stop codon transcription factor hit and run A in and out hit in 1. site-directed mutagenesis 2. 162

酶 TK run out intrachromosomal recombination FIAU gancyclovir A tag and exchange B FIAU gancyclovir 酶 recombinase knock in Cre/loxp FLP/frt Cre P1 FLP 34 bp A 2 13 bp inverted repeats loxp frt loxp frt loxp frt B loxp frt Cre FLP loxp frt C Cre/loxp FLP/frt D exon 2 Neo r 163

TK loxp frt exon 2 Cre FLP transfection Cre FLP FIAU loxp frt loxp frt exon 1 2 intron exon 2 gene knockout / Cre/loxp FLP/frt loxp frt DNA Cre plasmid transient transfection FIAU 1 3loxp frt 2 3loxp frt TK FIAU 1 2loxp frt TK FIAU -/- Cre transgenic mice Cre Cre Cre -/- 164

+/- Cre -/- Cre Cre Cre 1 3 loxp frt gene knockout Cre/loxp FLP/frt tet- tetracycline, tet doxycyclin dox Tet- A transactivator, tta tet repressor VP16 transactivation domain fusion protein tet dox tet dox tta Cre tet- tet-operatorcre tet dox tta Cre tet- Cre Cre loxp B tet rtta tet- tet dox tet- Cre Cre tet- Cre rearrangement germ line transmission 165

http://www.tbase.jax.org The Jackson Laboratory http://www.bioscience.org/knockout http://mkmd.bmn.com http://www.mshri.on.ca/develop/nagy/cre.htm Cre 166

(embryonic stem cell) (inner cell mass) A (blastocyst) (culture of embryonic stem cells) (electroporation) (homologous recombination) (+/-) B ( ) (wild type) (F1) (chimeric mice) (+/-) (F2) (+/-) (heterozygote) (-/-) (homoozygote) (+/-) (heterozygote) (+/+) (wild type) (A) (B) (A) (B) 167

DNA (A) (B) (A) (B) (*) 168

(A) (B) loxp/frt (C) loxp/frt Cre FLP Cre FLP Cre/loxp FLP/frt (A)loxp frt 34bp (B) loxp frt frt Cre FLP (C) loxp frt Cre FLP Cre/loxp FLP/frt FIAU Cre/loxp FLP/frt 169

(genomic locus) Cre/FLP FIAU (+/-) Cre Cre (-/-) (A) tet ----tet off tet dox tet dox tet -operator CMVmin tet -operator CMVmin (B) tet ----tet on tet dox tet dox tet -operator tet -operator CMVmin CMVmin (A) tet (B) tet 170

Doetschman, T., R. G. Gregg, N. Maeda, M. L. Hooper, D. W. Melton, S. Thompson and O. Smithies. 1987. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330:57657-57658. Evans, M. J., M. B. Carlton and A. P. Russ. 1997. Gene trapping and functional genomics. Trends Genet. 13:370-374. Hogan, B., R. Beddington, F. Costantini and E. Lacy. 1994. Manipulating the Mouse Embryo, 2 nd ed., pp. 253-290, Cold Spring Harbor Labotary, NY, USA. Le, Y. and B. Sauer. 2000. Conditional gene knockout using cre recombinase. Methods Mol. Biol. 136:477-485. Martin, G. R. 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA. 78:7634-7638. Muller, U. 1999. Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech. Dev. 82:3-21. Osada, J. and N. Maeda. 1998. Preparation of knockout mice. Methods Mol. Biol. 110:79-92. Ren, S. M. Li, H. Cai, S. Hudgins and P. A. Furth. 2001. A simplified method to prepare PCR template DNA for screening of transgenic and knockout mice. Contemp Top Lab Anim. Sci. 40:27-30. Thomas, K. R. and M. R. Capecchi. 1987. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51:503-512. 171

1980 1989 1990 gene targeting gene knockout Dolly totipotency 1997 Polly 30 60 172

fibroblast 2000 2002 α-1,3-20 22 112 115 2.2 1952 Briggs King 2n 1986 Willadsen 173

1989 Prather 1990 1997 Wilmut G0 -Dolly 1997 Schnieke -Polly 2000 Roslin Institute PPL 2000 3 5 α-1,3-2002 Lai PPL Dai 2002 3 Infigen Genmark 2 16 174

chimera 100 3 8 mm 36 48 60 80% 175

7 Regumate 20 mg/ 16 Regumate 1,500 2,000 IU pregnant mare's serum gonadotropin, PMSGPMSG 80 1,000 1,500 IU human chorionic gonadotropin, HCG PMSG HCGPMSG HCG 1,000 IU 500 IU HCG HCG 36 42 6 144 50% 10% 30% Park 2001 Lai 2002 α-1,3- PPL 176

squeeze aspiration 60 80% UV chemical enucleation cumulus cells granulosa cells embryonic stem cell-like 10% 37 39 5% CO 2 100% Wilmut 1997 G0 G1 maturation promotion factor, MPF 177

4 0.5% 100 200 1980 " fetal cells 2000 2002 3 Infigen Genmark Prairie State Semen Inc. "The man" "401K" 178

Infigen 1992 Chen 4 8 6 2 5 145.45% 199.36% 2001 1 11 795 11 55% 6/11 6 3 2002 2 15 87 4 14 7 12.14 10.30 100% 4 25 200 500 µg/ml 5 µg/ml 40 100 179

88 human decay accelerating factor, hdaf human leucocyte antigen class II, HLA-IIDR, DQ, and DP T- α- α- α-1,3-galactosyltransferase, α-gt α- α- α-gal 180

α-gt 1980 10% C 2000 Dolly PPL α-1,3-2002 Lai PPL Dai 181

1997 PPL 1998 4 13 telomere " " 182

1998 18 embryo cloning 183

" " " " 184

185

( ) 186

- 1 2 3 1 187

Betthauser, J., E. Forsberg, M. Augenstein, L. Childs, K. Eilertsen, J. Enos, T. Forsythe, P. Golueke, G. Jurgella, R. Koppang, T. Lesmeister, K. Mallon, G. Mell, P. Misica, M. Pace, M. Pfister-Genskow, N. Strelchenko, G. Voelker, S. Watt, S. Thompson and M. Bishop. 2000. Production of cloned pigs from in vitro systems. Nature Biotechnol. 18:1055-1059. Dai, Y., T. D. Vaught, J. Boone, S. H. Chen, C. J. Phelps, S. Ball, J. A. Monahan, P. M. Jobst, K. J. McCreath, A. E. Lamborn, J. L. Cowell-Lucero, K. D. Wells, A. Colman, I. A. Polejaeva and D. L. Ayares. 2002. Targeted disruption of the α1,3-galactosyltransferase gene in cloned pigs. Nature Biotechnol. 20:251-255. Lai, L., D. Kolber-Simonds, K. W. Park, H. T. Cheong, J. L. Greenstein, G. S. Im, M. Samuel, A. Bonk, A. Rieke, B. N. Day, C. N. Murphy, D. B. Carter, R. J. Hawley and R. S. Prather. 2002. Production of α-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295:1089-1092. Onishi, A., M. Iwamoto, T. Akita, S. Mikawa, K. Takeda, T. Awata, H. Hanada and A. C. F. Perry. 2000. Pig cloning by microinjection of fetal fibroblast nuclei. Science 289:1188-1190. Park, K. W., H. T. Cheong, L. Lai, G. S. Im, B. Kuhholzer, A. Bonk, M. Samuel, A. Rieke, B. N. Day, C. N. Murphy, D. B. Carter and R. S. Prather. 2001. Production of nuclear transfer-derived swine that expressed the enhanced green fluorescent protein. Anim. Biotechnol. 12:173-181. Polejaeva, I. A., S. H. Chen, T. D. Vaught, R. L. Page, J. Mullins, S. Ball, Y. Dai, J. Boone, S. Walker, D. L. Ayares, A. Colman and K. H. S. Campbell. 2000. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407:86-90. Prather, R. S., M. M. Sims and N. L. First. 1989. Nuclear transplantation in early pig embryos. Biol. Reprod. 41:414-418. Schnieke, A. E., A. J. Kind, W. A. Ritchie, K. Mycock, A. R. Scott, M. Ritchie, I. Wilmut, A. Colman and K. H. S. Campbell. 1997. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278:2130-2133. Wilmut, I., A. E. Schnieke, J. McWhir, A. J. Kind and K. H. S. Campbell. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385:810-813. 188

insertional recombination genome transformation carcinogensis concatamerization gene therapy DNA DNA cis-elements trans-acting factor 189

DNA recombination repair mutation gene expression DNA inversion deletion integration transposition oncogene homologous recombination 1 10 kb repetitive sequence ribosomal genes insertion sequences; IS prophage DNA 10 RecA RecBCD RuvA RuvB site-specific recombination λ transposition co-integrates DNA monomerization DNA DNA integrase, Int excisionase, Xis integration host factor; IHF FIS transpositional 190

recombination transposon DNA transposase target site 300 bp Alu 5% illegitimate recombination " nonhomologous " duplication DNA DNA DNA DNA B hepatitis B virus; HBV human papillomavirus; HPV DNA DNA DNA DNA position effect hfviii 191

DNA DNA DNA DNA DNA DNA DNA - A-T DNA Characterization Pronucleus ES cell-mediated Retroviral microinjection gene targeting infection Recombination Illegitimate Homologous Transpositional type recombination recombination recombination Frequency ca.10-4 ca.10-7 ca.1 Specificity None to low Very high Low to high Requirements None 5-10kb sequences Entry in retrovirion homology specific LTR sequence at both ends Effects on host Random deletion, None to minimal 4-6bp duplication at target rearrangement integration site Effect on Random deletion, None to minimal 2bp lost from each inserted DNA duplication, and end rearrangement 192

Southern blot hybridization 2 10 A HPV18/LCR SV40/TAg DNA slot-blot 18LT-12 18LT-28 18LT-52 2 4 18LT-11 18LT-22 200 280 DNA 18LT-SV40TAg A αla-plf B αla-hfviii C - H-T tandem repeat H-H T-T inverted repeats HPV LCR 40 SV40 TAg A 12 XbaI SmaI DNA 5'- 3'- 36 cis-element LCR/TAg PCR PCR 6% DNA 36 31 193

5 3'-5' 3'-3' 5'-5' α β γ * DNA 18LT31α4/β2 DNA 6 3'-5' 25% internal deletion 91.7% 18LT-28/α1 18LT-31/β2 18LT-11/α6 3'-SmaI 5'-XbaI 31 62 3'-SmaI 5'-XbaI 16.7% vs 15.4% DNA 1 200 bp 3'- 80.1% vs 61.6% 200 bp 5'- 3'- 23.1% vs 3.3% 5 overlapping 61.3% 19/31 1 6 "TCCC" DNA 194

Transgenic Transgene Nucleotide deletion a Insertion mouse junction Left tg Overlapping Right tg Total sequnce (bp) sequnce (bp) (bp) ( )3-5 junction ( type): 18LT-28 1 0 -- 0 0 -- 18LT-28 2 0 -- 0 0 ATCT 18LT-31 1 4 TGGATT 37 47 -- 18LT-31 2 1 TCCC 8 13 -- 18LT-31 3 5 -- 10 15 -- 18LT-31 4 144 -- 3715 3859 ACCTAA 18LT-31 5 I.d. GT I.d. 480 -- 18LT-31 6 1 TCCC 8 13 -- 18LT-22 1 93 C 9 103 -- 18LT-22 2 44 TGTGG 35 84 -- 18LT-22 3 1 TCCC 8 13 -- 18LT-22 4 3 -- 12 15 TT 18LT-22 5 94 GCAA 122 222 -- 18LT-22 6-1 122 -- 287 409 -- 18LT-22 6-2 I.d. -- I.d. 313 -- 18LT-22 6-3 97 -- 122 219 -- 18LT-11 1 188 -- 0 188 CGACT 18LT-11 2 1 TCCC 8 13 -- 18LT-11 3 5 GGAT 5 14 -- 18LT-11 4 125 TTGC 3515 3644 -- 18LT-11 5 105 AC 414 523 -- 18LT-11 6 0 -- 0 0 CGACT ( ) 3-3 junctions ( type): 18LT-2 1 76 ACAAA 38 119 -- 18LT-31 1 97 CA 8 109 -- 18LT-31 2 0 -- 0 0 -- 18LT-22 1 138 TTA 3702 3843 -- 18LT-22 2 122 CA 8 134 -- 18LT-11 1 154 -- 8 162 -- ( ) 5-5 junctions ( type): 18LT-31 1 91 ATT 2929 3033 -- 18LT-22 1 7 GAT 15 25 -- 18LT-11 1 93 AAA 3412 3508 -- ( ) Transgene-cellular junctions ( type): 18LT-22 1 41 *******Cellular sequence******* 18LT-22 2 3434 *******Cellular sequence******* 18LT-22 3 3433 *******Cellular sequence******* 18LT-11 1 3236 *******Cellular sequence******* 18LT-11 2 171 *******Cellular sequence******* a In this column, deletion sizes in the adjoining left and right transgene (tg) are shown. I.d. indicates internal deletion within transgene. - indicates absence of sequence. insertion sequences, IS 16.1% DNA " " 195

DNA DNA motif DNA DNA DNA motif 10 DNA motif topoi 'CTY'; 'GTT' Homo-R/-Y - Alternating R-Y A-T A-T rich motifs 5 36 72 topoi motifs 97.2% topoi motifs topoi motifs topoi* 62.5% 20 A-T DNA motifs 54.2% 36.1% 29.2% topoi* motif motifs Homo-R/-Y topoi* motif 73.3% topoi motif topoi DNA Homo-R/-Y DNA S1 enhancer sequences DNA motifs 3.7 kb DNA motifs R/Y A-T Homo-R/-Y motif 5' 5' Homo-R/-Y motif 18 bp 27 bp 5'-XbaI B- DNA DNA Homo-R/-Y motif 196

hypervariable minisatelite 10 DNA motifs No. of seq. with motif Motif / no. analyzed Frequency (%) (A) Single motif: Topo motifs 70/72 97.2 Topo * motifs 45/72 62.5 Homo-R or homo-y 39/72 54.2 Alternating R-Y 26/72 36.1 (A-T)-rich 21/72 29.2 (B) Motifs coincidental with Topo * sites : Homo-R or-y/topo * 33/45 73.3 Alternating R-Y/Topo * 14/45 31.1 (A-T)-rich/Topo * 14/45 31.1 Homo-R or-y/alt R-Y/Topo * 9/45 20.0 Homo-R or-y/(a-t)-rich/topo * 8/45 17.8 12 LCR/TAg 2 280 36 7:3 DNA DNA 197

DNA DNA 1 6 topo I motif B DNA A-T topoi unwiding DNA DNA 1 6 DNA DNA A DNA topoi DNA motifs Homo-R/-Y motifs 198

(A) HPV18- LCR/SV40-TAg ( ) BglI Southern blot ( ) (B) (C) ala-plf ala-hfviii Southern blot (A) 18LT-22 β2 (B) 199

Chen, C. M., C. H. Wang, S. C. Wu, C. C. Lin, S. H. Lin and W. T. K. Cheng. 2002. Temporal and spatial expression of biologically active human factor VIII in the milk of transgenic mice driven by bovine α-lactalbumin promoter. Transgenic Res. 11:257-268. Chen, C. M., W. T. K. Cheng and K. B. Choo. 1995. Frequent deletions and sequence aberrations at the transgene junctions of transgenic mice carrying the papillomavirus regulatory and the SV40 TAg gene sequences. Transgenic Res. 4:52-59. Derbyshire, M. K., L. H. Epstein, C. S. Young, P. L. Munz and R. Fishel. 1994. Nonhomologous recombination in human cells. Mol. Cell. Biol. 14:156-169. Eisenstark, A. 1977. Genetic recombination in bacteria. Ann. Rev. Genet. 11:369-396. Holliday, R. 1964. A mechanism for gene conversion in fungi. Genet. Res. 5:282-304. Konopka, A. K. 1988. Compilation of DNA strand exchange sites for non-homologous recombination in somatic cells. Nucleic Acids Res. 16:1739-1758. Moser, H. E. and P. B. Dervan. 1987. Sequence-specific cleavage of double helical DNA by triple helix formation. Science 238:645-650. Nicolas, A. L. and C. S. Young. 1994. Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific. Mol. Cell. Biol. 14:170-180. Pfeiffer, P., S. Thode, J. Hancke and W. Vielmetter. 1994. Mechanisms of overlap formation in nonhomlogous DNA end joining. Mol. Cell. Biol. 14:888-895. Roth, D. B., G. N. Proctor, L. K. Stewart and J. H. Wilson. 1991. Oligonucleotide capture during end joining in mammalian cells. Nucleic Acids Res. 19:7201-7205. 200

DNA DNA DNA DNA phenol chloroform ether low melting point agarose ion-exchange resin chromatography column DNADNA DNA DNA DNA 201

DNA 1 2 ng/µl DNA 2 ng/µl 10 ng/µl 0.5 1.0 2.0 ng/µl DNA 2 1.0 2.0 ng/µl 0.5 ng/µl DNA holding pipette injection needle 30 80 µm 20 µm 4 µm DNA DNA DNA DNA DNA DNA 202

DNA lacz DNA lacz X-gal X-gal DNA X-gal lacz ( ) DNA Southern blot hybridization DNA regulator sequence structural gene intron exon splicing 203

1. cdna 2. cdna 3 3. DNA DNA cdna DNA cdna DNA whey acid protein, WAP 1 gene targeting 204

DNA DNA methylation DNA 5' DNA DNA DNA DNA 5' DNA RNA DNA β- 5' β- 3' β- 3' 3' 99.2% 72.9 98.6% β- mrna β- mrna 205

5' CoRE composite response element CoRE 5' -DNA - NF1 nuclear factor 1 STAT5 signal transducers and activators of transcription 5 β- STAT5 C/EBP β CCAAT/enhancer binding protein PMF pregnancy-specific mammary nuclear factoryy1 Yin Yang nuclear factor 1 OCT1 octamer binding protein 1 1. NF1 NF1 DNA palindrome 5 -PyTGG(N)7-CCAPu-3 NF1 β- 3 NF1 NF1 NF1 CoRE NF1 NF1 DNA β- 5-149 -406 5 NFI NF1 2. STAT5 STAT5 5 -ACTTCTTGGAATT- 3 5 -TTCTTGGAA-3 STAT5 DNA mammary gland factor, MGF milk protein binding factor, MPBF STAT5 STAT5a, b 93% 6 MGF 21 STAT5a, b STAT5 206

STAT5 STAT5 DNA STAT5 3. C/EBP C/EBP 7 β- -240-824 C/EBP 3 β- C/EBPβ LAP liver-enriched activating protein LIP liver-enriched inhibitory protein LIP transactivation domain LAP LIP LAP C/EBP heterodimer LAP/LIP C/EBP 4. PMF 5' PMF -11 +7; -366-347 5 -TGAT/ATCA-3 PMF β- PMF 5. YY1 YY1 β- α- γ- 5' YY1 electrophoretic gel mobility shift assay YY1 β- 5' YY1 YY1 STAT5 STAT5 YY1 DNA YY1 β- 207

β- 5' - C/EBPb YY1 β- 6. OCT1 αs2-5' OCT1-50 -210-230 -260-480 5' -50 OCT1 7. 208

A B (A) (B) DNA ( ) DNA (A) (B) A B C TK/lacZ X-gal lacz (A B) ; 11 X-gal lacz (C) : : 209

1999 47:101-105 1988 : 37:97-110 2001 : 30:45-48 Brink M. F., M. D. Bishop and F. R. Pieper. 2000. Developing efficient strategies for the generation of transgenic cattle which produce biopharmaceuticals in milk. Theriogenology 53:139-48. Houdebine L. M. 2002. The methods to generate transgenic animals and to control transgene expression. J. Biotechnol. 98:145-160. Houdebine L. M., S. Rival, T. Pantano, G. Jolivet, D. Thepot and J. Attal. 2002. Transgenesis for the study and the control of lactation. Reprod. Nutr. Dev. 42:117-125. Huang, M. C. and J. S. Chao. 2001. Regulatory sequences of the 5' flanking region of goat β-casein gene. AJAS 14:1628-1633. Pintado B. and A. Gutierrez-Adan. 1999. Transgenesis in large domestic species: future development for milk modification. Reprod. Nutr. Dev. 39:535-544. Salaheddine M., E. Thole and M. Brink. 2002.Transgenic cattle constitute a breakthrough in production of innovative medicine. From milk to medicine. Tijdschr Diergeneeskd. 127:226-230. van Berkel P. H., M. M. Welling, M. Geerts, H. A. van Veen, B. Ravensbergen, M. Salaheddine, E. K. Pauwels, F. Pieper, J. H. Nuijens and P. H. Nibbering. 2002. Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nat. Biotechnol. 20:484-487. 210

1985 (mice) (chickens) (salmon) (rats) (Japanese quail) (trout) (rabbits) (Tilapia) (cattles) (carp) (pigs) (catfish) (sheep) Medaka (goats) (zebrafish) (monkey) (loach) (goldfish) (pike) pronuclear microinjection sperm vector embryonic stem cells, ES cells nuclear transfer transfection 211

bioreactor growth hormone insulin like growth factor-1, IGF-1 myostatin 1982 Palmiter Hammer 1985 cdna metallothionein MT promoter 1989 Pursel synovitis reduced fertility insulin resistance gastric ulcer dermatitis nephritis pneumonia cdna MT 5 11 growth hormone-release factor; GHRF GHRH 212

GHRH IGF-1 IGF-1 DNA IGF-1 Pursel IGF-1 α-actin promoter IGF-1 IGF-1 IGF-1 20 60 daily weight gain body composition60 IGF-1 negative regulator 1997 Lee myostatin myostatin knockout mouse myostatin 86% 5% lactose4.1% 3.6% 0.7% 1996 Gutierrez-Adan κ-casein 213

micelle α-lactalbumin 1999 lactase lactoferrin, LF amino acid cysteine keratin Cysteine serine cysteine serine acetyltransferase CysE ο- acetylserine sulphydrase CysM MT CysE CysM IGF-1 immunoglobulin 214

Mx1 influenza virus Mx1 Mx1 mrna mastitis lysostaphin endopeptidase lysostaphin lysostaphin interferon Chen 1988 β-interferon Salter Crittenden 1989 Avian Leukosis Virus ALV bioreactor whey acidic protein WAP α-lactalbumin α-lactoglobulin β-casein α-antitrypsin ATT ATT 35 g ATT ATT 215

α-antitrypsin Emphysema/cystic fibrosis PPL α-glucosidase Glycogen metabolic disease Pharming Antibodies Anticancer CellGenesys, Genzyme Antithrombin III Thromboses Genzyme Collagen Rheumatoid arthritis Pharming CFTR Cystic fibrosis Genzyme Factor IX Haemophilia Genzyme, PPL Protein C Blood Coagulation ARC, PPL Haemoglobin Blood substitute Baxter Lactoferrin Infant formula Pharming Fibrinogen Tissue sealant development ACR, PPL Albumin Trauma/burn treatment Pharming TPA Dissolve fibrin clots Genzyme xenotransplantation hyper-acute rejection humanization White 1992 human decay accelerating factor, hdaf 1995 hdaf HLA 2002 216

1997 Wilmut nuclear transfer 217

Bawden, C. S., A. V. Sivaprasad, S. K. Verma, S. K. Walker and G. E. Rogers. 1995. Expression of bacterial cysteine biosynthesis genes in transgenic mice and sheep: toward a new in vivo amino acid biosynthesis pathway and improved wool growth. Transgenic Res. 4:87-104. Brophy, B., G. Smolenski, T. Wheeler, D. Wells, P. L Huillier and G. Laible. 2003. Cloned transgenic cattle produce milk with higher levels of β-casein and κ-casein. Nat. Biotechnol. 21:157-162. Devlin, R. H., T. Y. Yesaki, E. M. Donaldson, S. J. Du and C. L. Hew. 1995. Production of germline transgenic Pacific salmonids with dramatically increased growth performance. Can. J. Fish Aquat. Sci. 52:1376-1384. Gutierrez-Adan, A., E. A. Maga, H. M. Meade, C. F. Shoemaker, J. F. Medrano, G. B. Anderson and J. D. Murray. 1996. Alteration of physical characteristics of milk from bovine kappa-casein transgenic mice. J. Dairy Sci. 79:791-799. Lavitrano, M., M. L. Bacci, M. Forni, D. Lazzereschi, C. D. Stefano, D. Fioretti, P. Giancotti. G. Marfe, L. Pucci, L. Renzi, H. Wang, A. Stoppcciaro, G. Stassi, M. Sargiacomo, P. Sinibaldi, V. Turchi, R. Giovannon, G. D. Casa, E. Seren and G. Rossi. 2002. Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hdaf) transgenic pigs for xenotransplantation. Proc. Natl. Acad. Sci. USA 99:14230-14235. Müller, M., B. Brenig, E. L. Winnacker and G. Brem. 1992. Transgenic pigs carring cdna copies encoding the murine Mx1 protein which confers resistance to influenza virus infection. Gene 121:263-270. Palmiter, R. D., R. L. Brinster, R. E. Hammer, M. E. Trumbauer, M. G. Rosenfeld, N. C. Birnberg and R. M. Evans. 1982. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth fusion gene. Nature 300:611-615. Pinkert, C. A. 1997. The history and theory of transgenic animals. Lab. Anim. 26:29-34. Tu, C. F., K. Tsuji, K. H. Lee, R. Chu, T. J. Sun, Y. C. Lee, C. N. Weng, C. J. Lee. 1999. Generation of HLA-DP transgenic pigs for study of xenotransplantation. Int. Surg. 84:176-182. Wilmut, I., A. E. Schneike, J. McWhir, A. J. Kind and K. H. Campbell. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385:810-813. 218

1 2 2 1 2 -gene knockout nuclear transfer -transgenic technology cellular commitment oocyte reprogram haematopoietic stem cell 219

post genome world 1985 15 30 50% embryonic stem ES cell 220

1. - ENU 1 α-synuclein α-synuclein neocortex hippocampus substantia nigra α-synuclein 2 SOD1 superoxide dismutase 1 NFs neurofilaments Amyotrophic lateral sclerosis; ALS SOD1 NF 221

SOD1 NF 3 renin angiotensinogen renin angiotensinogen 4 lox P A mttfa; mitochondrial transcription factor A Cre A Cre creatinine kinase 13 mitochondrial cardiomyopathy Cre 2. - random integration transgenesis targeted modification of genes and genome gene knockout introducing subtle changes into a disease gene conditional gene targeting inducible conditional gene targeting large deletion/inversions ENU 222

1 sickle cell disease β globin S S SAD α2-globin - α 2 -SADSAD SAD Agamma globin α β-sad Agamma 9 16% SAD SAD 2 retinal degeneration Bcl-2 Bcl-2 Bcl-2 3 cystic fibrosis, CF 20 cystic fibrosis transmembrane conductance regulator, CFTR 223

4 retinitis pigmentosa RP Rhodopsin rod photpreceptor Rhodopsin RP 3. transgenic mutagenesis homology searching cdna proto-oncogene growth factor receptor C. elegans Xenopus ENU 224

1 ENU ENU mutagenesis screens ENU N-ethyl-N-nitrosourea 苷 ethyl DNA DNA 6 10-3 175 1 1000 1 100 F1 F1 founder F2 F2 F1 founder backcross F3 2 gene trap ES 225

ES ES splice acceptor site reporter gene selectable marker A. enhancer trap lacz ES EBs embryonic bodies Southern blot hybridization B. promoter trap intron lacz null hypomorph 226

dominant negative alleles ENU 1. 1 α1-antitrypsin 227

reticulocyte 2 gonadotropins uroplakin II hgh hgh 0.1 0.5 mg/ml 3 P12 hgh 0.5 mg/ml 4 1999 5 DNA Baculovirus Bac 6 IGF1 NGF-β hgh erythropoietin thrombopoietin 228

parathyroid hormone fibrinogen collagenextracellular superoxide dismutase 2. 1 microinjection DNA GFP fluorescent in situ hybridization DNA DNA episomal vector linear artificial chromosome 2 cell transfection embryo cloning 1997 homologous recombination 229

3 generation of chimeric animals from multipotent cells ES EG 1999 EG 4 gene transfer into sperms oocytes 3. DNA DNA, position effect centromere telomere 1 RNA 2 ectopic expression enhancer 1 A. 230

codonb. AUG Kozak C. N signal peptide D. cdna E. mrna 5 5 -untranslated region IRES 2 A. promoter WAP whey acidic protein β-lactoglobulin β-lactoglobulin B. the distal regulatory elements multicopies LCR locus control region DNA DNA 100 300 kb insulator Insulator DNA insulator 5 HS4 chromatin opener 5 HS4 231

MAR matrix attached region nuclear matrix 3 DNA β-casein αs1-casein 4. 1 glycosylation human antithrombin III sialylation lipase α1-antitrypsin EC superoxide dismutase C C carboxylation 2 hgh 232

WAP EC superoxide dismutase 3 prion hyperacute rejection; HAR α-gal hdaf CD59 MCP 3 acute vascular rejection; AVR antiapoptotic gene 5 7 10% 100 10 kg 233

CHO IgA 24 ml 20 µg IgA peptide erythropoietin 5 expression vector 234

1 ml ScFv Fab Fab IgA IgA α-lactalbumin prion [ 1 4 ] 4 20 180 cm 235

236

ES ES (3) (2) (4) (1) 237

Abuin, A., K. H. Holt, K. A. Platt, A. T. Sands and B. P. Zambrowicz. 2002. Full-speed mammalian genetics: in vivo target validation in the drug discovery process. Trends Biotechnol. 20:36-42. Cecconi, F. and B. I. Meyer. 2000. Gene trap: a way to identify novel genes and unravel their biological function. FEBS Lett. 480:63-71. Cozzi, E., B. Soin, B. Holmes and D. White. 2000. Genetic engineering of the donor as an approach to clinical xenotransplantation. Transplant. Proc. 32:2701-2703. Hardouin, S. N. and A. Nagy. 2000. Mouse models for human disease. Clin. Genet. 57:237-244. Houdebine, L. M. 2000. Transgenic animal bioreactors. Transgenic Res. 9:305-320. Larrick, J. W. and D. W. Thomas. 2001. Producing proteins in transgenic plants and animals. Curr. Opin. Biotechnol. 12:411-418. Lubon, H. and C. Palmer. 2000. Transgenic animal bioreactors-where we are. Transgenic Res. 9:301-304. McWhir, J. 2000. A second generation of animal models. Transgenic Res. 9:345-346. Petters, R. M. and R. Jeffrey. 2000. Sommer Transgenic animals as models for human disease. Transgenic Res. 9:347-351. Wheeler, M. B. and E. M. Walters. 2001. Transgenic technology and applications in swine. Theriogenology 56:1345-1369. 238

gametes embryo 1972 1 / -80 1972 0 0 239

dulbecco s phosphate-buffered saline, DPBS 300 mosm/kg NaCl PBS NaCl 400 mosm/kg PBS NaCl 200 mosm/kg 80% 90% 99% NaCl KCl salty -3 supercooling -10 240

seeding PBS PBS -6 0.3 / -25-60 4 1. 2. 3. 4. 241

DMSO ethylene glycol 1,2-propandiol sucrose raffinose lipoproteins DMSO DMSO 1 2 M 242

3 10 5 10 1.2 M 1.0 M 0.8 M cryopreservation freezing 2 3 recrystallization 243

4 1.5 M DMSO -50-80 2-4 M DMSO 0.25 M sucrose vitrification 1.5 M DMSO 1.5 M 1,2-propandiol -35 GV-stage 3.5 M DMSO 0.5 M sucrose 0.1 1.0 / phase hypertonic organelles 244

-30-40 25 37 215 450 / -60-110 4 25 / -78-196 1.5 M EG Ethylene Glycol0.4% BSA 0.1 M sucrose DPBS 245

EG 6 1. 1.5 M EG 2. 3. 6-9 -6 4. -6 10 5. 0.5-32 6. 1. 2. 5 3. 25 4. 5. Agca, Y., R. L. Monson, D. L. Northey, O. A. Mazni, D. M. Schaefer and I. J. Rutledge. 1998. Transfer of fresh and cryopreserved IVP bovine embryos: Normal calving, birth weight and gestation lengths. Theriogenology 50:147-162. Eppig, J. J. and M. J. O Brien. 1996. Development in vitro of mouse oocytes from primordial follicles. Biol. Reprod. 54:197-207. Lehn-Jensen, H. and S. M. Willadsen. 1983. Deep-freezing of cow half and quarter embryos. Theriogenology 19:49-54. Leibo, S. P. and N. M. Loskutoff. 1993. Cryobiology of in vitro-derived bovine embryos. Theriogenology 39:81-94. Moore, N. W., T. D. Heath, R. P. Elsden, G. E. Siedel. Jr, B. W. Pickett, T. Takeda, A. B. Ferguson and S. J. Miller. Bovine embryo transfer workshop. 1984. The University of Sydney: Proceedings No. 70. Pedersen, R. A., A. McLaren and N. L. First. 1991. Animal applications of research in mammalian development. Cold Spring Harbor Laboratory Press, NY, USA Pugh, P. A., A. E. L. Ankersmit, L. T. McGowan and H. R. Tervit. 1998. Cryopreservation of in vitro produced bovine embryos: 246

Effects of protein type and conception during freezing or liposomes during culture on post-thaw survival. Theriogenology 50:495-506. Telfer, E. E. 1998. In vitro models for oocyte development. Theriogenology 49:451-460. Wright, R. W. Jr. and J. Ellington. 1995. Morphological and physiological differences between in vivo- and in vitro-produced preimplantation embryos from livestock species. Theriogenology 44:1167-1189. 247

tpa urokinase CD4 FIX α 1 antitrypsin α 1 AT lactoferrin, LF tpa antithrombin III ATIII FIX α 1 AT protein C IL-2 tpa precipitation 248

1. ph 2. 3. 4. 5. 1. 2. ph ph isoelectric point, pi ph pi NH + 4, SO 2-4 gel filtration 249

external volume Ve internal volume Vi (Ve) (Vi) 1. 2. 3. resolution column length elution time column length1. 2. 3. eluent 0.15 N 4. linear flow ratecm/hr 250

volume flow rateml/min (volume flow rate: cm 3 /min) 60 (cm 2 ) Affinity Chromatography matrix ligand elution 1. hydrophilic 2. large pores 3.rigid 4. inert 5. stability matrix dextran agarose polyacrylamide ligand group matrix K d 10-4 10-8 M K d 10-4 M K d 10-8 M ligand matrix spacer arm 251

Ligand Lectins Poly U Poly A Protein A Lysine Benzamidine Heparin specificity binding molecules Polysaccharides Poly A Poly U Fc antibody rrna dsdna plasminogen Serine proteases Lipoproteins, DNA, RNA ionic exchange chromatography ampholyte lysine arginine aspartic acid glutamic acid ph ph stationary phase mobile phase counterion separation binding elution retention electrostatic interaction binding elution 252

cellulose dextran agarose polystyrene DEAE diethyl-aminoethyl CM carboxymethyl DEAE CM - SP -CH 2 CH 2 CH 2 SO 3 CM -OCH 2 COO - Q -N + (CH 3 ) 3 DEAE -N + (C 2 H 5 ) 2 H ph isoelectric point, pi ph pi ph pi ph pi ph pi ph electrophoretic titration curve 253

ph ph ph 10 mm < 5 ms/cm ph Counterion pka 25 Acetate Na + 50 mm 4.8 BICINE Na + 50 mm 1.8, 8.4 Citrate Na + 20 mm 3.1, 4.8, 6.4 Formate Na + 50 mm 3.8 HEPES Na + 50 mm 3, 7.6 Malonate Na + 50 mm 5.7 Phosphate Na + 50 mm 2.1, 7.2, 12.3 bis-tris Cl - 20 mm 6.5 1,3-diaminopropane Cl - 20 mm 10.5 Diethanolamine Cl - 20 mm 8.9 Ethanolamine Cl - 20 mm 9.5 Triethanolamine Cl - 20 mm 7.8 Tris Cl - 20 mm 8.1 BICINE, N,N-bis(2-hydroxyethyl)glycine bis-tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane HEPES, N-(2-hydroxyethyl)piperazine-N -(2-ethanesulfonic acid) 254

counterion - structure-function studies enzymology cellular location - tissue-organ specific gene expression functional genomics 1. 2.. 3. SDS- SDS-polyacrylamide gel electrophoresis, SDS-PAGE SDS sodium dodecyl sulfate SDS SDS-PAGE SDS-PAGE denature 255

ph ph PI ph 8.3 ph pi 8.3 PAGE polyacrylamide gel electrophoresis SDS PAGE PVDF blotting N- C- N- dansylation dansyl HCl N PTH NH 2 NH 2 Western blot hybridization N- C- GST His Taq HPLC IEF SDS-PAGE 256

bovine serum albumin, BSA immunoglobin UV Biuret method BCA bicinchoninic acid method Lowry method Bio-Rad Protein Assay UV ultraviolet absorption method UV 280 nm 200 nm 280 nm 200 nm phenylalanine tryptophan histidine tyrosine 280 nm 257

peptide bond 210 nm C O 200 nm C O C C colorimetric assays 1. Biuret method C O C O biuret ammonium persulfate Tris 2. Lowry method Biuret method C O Folin-Ciocalteau phosphomolybdic-phosphotungstate 700 nm 500 nm Biuret method 0.1 mg Foloin reagent ammonium persulfate thiol 3. BCA bicinchoninic acid method 258

Lowry method Cu 2+ Cu + BCA Folin-Ciocalteau Cu + 562 nm Folin-Ciocalteau 4. Bio-Rad protein assay coomassie brilliant blue G-250 465 nm 595 nm arginine BSA 1.0 10.0 µg/ml immunoglobin 1.2 25 µg/ml microtiter plate assay BSA BSA immunoglobin G ph HCl NaOH ph EDTA CaCl 2 EDTA 259

--- 4 6 8 10 ph ph 260

Citric acid Lactic acid Butanedioic acid Acetic acid Malonic acid MES Phosphate HEPES 2.5 3 4 5 6 7 8 9 BICINE ph N-methylpiperazine Piperazine bis-tris bis-trispropane Triethanolamine Tris N-methyldiethanolamine 1,3-diaminopropane ethanolamine Piperazine 1,3-diaminopropane Piperazine 4.5 5 6 7 8 9 10 1 1 12 ph 261

SDS- PAGE N- 262

Duke, T. 1998. Separation techniques. Curr. Opin. Chem. Biol. 2:592-596. Griffin, T. J., D. R. Goodlett and R. Aebersold. 2001. Advances in proteome analysis by mass spectrometry. Curr. Opin. Biotechnol. 12:607-612. Hennion, M. C. 1999. Solid-phase extraction: method development, sorbents, and coupling with liquid chromatography. J. Chromatography 856:3-54. Holthuis, J. J. and R. J. Driebergen. 1995. Chromatographic techniques for the characterization of proteins. Pharm. Biotechnol. 7:243-299. Kellogg, D. R. and D. Moazed. 2002. Protein and immunoaffinity purification of multiprotein complexes. Methods Enzymol. 351:172-183. Murray, P. D. 1992. Methods in Enzymology, Vol. 182, Guide to protein purification, pp. 50-68, 285-390. Academic, California, USA. Ong, S. E. and A. Pandey. 2001. An evaluation of the use of two-dimensional gel electrophoresis in proteomics. Biomol. Eng. 18:195-205. Pharmacia, 1991. Gel filtration, Principles and Methods, pp. 6-13. Rahms I Lund, Sweden. Porath, J. 1997. From gel filtration to adsorptive size exclusion. J. Protein. Chem. 16:463-468. Sii, D. and A. Sadana. 1991. Bioseparation using affinity techniques. J. Biotechnol. 19:83-98. Thomas, J. G. and L. O. Dale. 1994. Concepts in Protein Engineering and Design, pp. 47-107, Walter de Gruyter & Co., Germany. 263

- 21 87 11 90 7 89 264

exogenous gene endogenous gene biohazard 1. transgene 2. 3. insertional mutation, im mobile elements viral receptor prion viral vector adenovirus retrovirus insulators 1. risks for animal and human health 1 / toxicity and food quality/safety 265

bioavailablity 2 xenotransplantation genome prion 3 4 drug resistance gene marker 2. risks for the environment unpredictable gene expression or transgene instability 3. risks for agriculture 266

French Commission of Biorisk Evaluation 3R- reduction refinement replacement cystic fibrosis, CF genotype phenotype gnotobiotic 267

homozygote Guide for the Care and Use of Laboratory Animal, Animal Welfare Act, NIH Guideline for Research Involving Recombinant DNA Molecules The Canadian Council on Animal Care CCAC CCAC Guideline on Transgenic Animals CCAC statement Ethics of Animal Investigation Directive 86/609/EEC ECVAM European Center for the Validation of Alternative Methods -The Use of Transgenic Animals in the European Union 1. 1 barrier specific pathogen free, SPF barrier high-efficiency particulate filter micro-isolator individual ventilated cage, IVC trexler-type isolator 2 Guide for the Care and Use of Laboratory Animal 268

standard operation procedure, SOP 3 sentinel animal Mycoplasma pulmonis Mouse hepatitis virus MHV Sendai virus Ectromelia Virus Hanta virus, Minute Virus of Mice MVM Clostridium piliformis, Pneumonia Virus of Mice PVM Theiler s Encephalomyelitis Virus TEMVLymphocytic Choriomeningitis Virus LCMV Rotavirus Reovirus Type 3 Reo-3Mouse Adenovirus Mouse K Virus pin-worm fur-mite 2. 269

barrier facility embryo transfer 3. C57BL/6 FVB 4. transmissible spongiform encephalopathy TSE 5. 100 250 6. 30 45 cm 270

FDA CCAC recapture 7. 8. 9. 1 biohazard CDC/NIH Biosafety in Microbiological and Biological Laboratories NIH Guideline for Research Involving Recombinant DNA Molecules 2 identification ear tag cage card 271

3 10. refinement 1. 1 2 DNA. 3 4 phenotype 272

5 6 standard operation procedure, SOP 7 end point Transgenic Sheet Information Animal Care Committee Institutional Biohazard Committee 2. 3. end point 1 2 3 4 CO 2 273

4. 1 2 3 4 5 6 5. 10 6. ILAR 1992 interspecies committee 1. transgene TgX(YYYYY)#####Zzz 1 TgX 274

Tg X H R N H homologous recombination N nonhomologous insertion R retrovirus microinjecion TgN MuLV vector TgR ES cells TgH 2 YYYYY 6 11 3 #####Zzz Zzz ILAR ##### 5 1 99,999 #####Zzz 11 2. 1 C57BL/6J-TgN(CD8GEN)23Jwg CD8 CD8GEN C57BL/6JJon W. Gordon Jwg non-homologous insertion 275

23 founder 2 Crl:ICR-TgN(SVDhfr)432Jwg mouse dihydrofolate reductase Dhfr gene SV40 early promotor4 kb Jon W. Gordon Jwg 32 founder Charles River Laboratories Crl ICR 3 TgN(GPDHim)Bir glycerol phosphate dehydrogenase GPDH C57BL/6J SJL/J insertional mutation, im Birkenmeier Bir 4 129/J-TgH(SV40Tk)65Rpw SV40-thymidine kinase 129/J Richard P. Woychik Rpw 65 1. FVB/N C57BL/6 F1 F1 hybrid CB6 F1 BALB/c C57BL/6 DB6 F1 DBA/2 C57BL/6 outbred stock homozygous founder 1 intercross 2 backcross F1 276

10 2. 1 founder 2 founderc57bl/6 C57BL/6 99% C57BL/6 C57BL/6 congenic strain C57BL/6 C57BL/6 congenic strain 129 C57BL/6 129 129 C57BL/6 2.5 3 congenic strain congenic strain microsatellite simple sequence length polymorphisms SSLP nucleotide repeats PCR primer pairs C57BL/6 3 4 1 2 C57BL/6 congenic strain 21 WTO 277

278

279 congenic strain

2001 2000 pp. 37-39 1999 2 Animal Welfare Act. 2002. USDA:http://www.aphis.usda.gov/oa/ pubs/awact.html Genetic engineering: animal welfare and ethics. 1999. A discussion paper from the Boyd Group. Guidelines for Research Involving Recombinant DNA Molecules. 1999. NIH Guidelines. Hessler, J. R., J. R. Broderson and C. S. King. 1999. Rodent quarantine: facility design and equipment for small animal containment facilities. Lab. Animal 28:34-40. Joyner, A. L. 1999. Gene Targeting: a Practical Approach. Oxford press. Marie, B. 1997. Transgenic Animals, Animal Welfare and Ethics. Canadian Council on Animal Care (CCAC). Marie, H. L. 2000. Transgenic Animals: Generation and Use. pp. 559-562. Harwood Academic Publishers. Muriel, T. D. 1997. Guidelines for Naming Transgenic Mice. Lab. Animal. The Report and Recommendations of ECVAM Workshop 28 1,2. 1998. The Use of Transgenic Animals in the Europe Union. ATLA 26:21-43. 280

pp. 37-39 DNA DNA P1 P2 1. LS-1 LS-1 DNA 2. 1. 1. 1 DNA 2 2. 1 A. DNA DNA DNA B. DNA DNA a b 281

c d 2 DNA DNA 3 A. B. a b c DNA d e f g h i j 282

1. 2. 3. 283

DNA - (P2) (P1) 1 - (1) (2) (3) (P3) (P2) (P1) 1 -(1) 2 2 2 2 2 ( -(3) P2 P1 P3 P2 P1 ) [ ] (P2 P1 P3 P2 P1 ) 1. 2. ( ) 1-1 - 284

1. Heterozygotes Homozygote 2. 3. 4. 5. 6. 7. 8. 9. 10. 285

pregnant mare s serum gonadotropin, PMSG human chorionic gonadotropin, HCG HCG 10 13 LH PMSG 15 20 HCG LH PMSG HCG 42 48 vaginal plug 3.5 12h 4 8 8 20 PMSG HCG 1 ml 26G 1/2-inch 70% M16 medium 1 ml M2 medium 1 ml 1 ml30g 1 ml 90 mm petri dish Pasteur pipette hyaluronidase 286

5 10 IU PMSG a, b 44 48 HCG 5 10 IU/ HCG c 1. 2. 3. d M2 medium 1. A. M2 medium 30G a B. M2 medium 2. A. M2 medium 26G b B. M2 medium C. M2 medium c 2d 2 3 e M2 medium 3 6 fm16 medium 5% CO 2, 37 287

a b c d a, b c d a b c d e f a 30G M2 medium b 26G M2 medium c M2 medium d 0.5 e 2-3min f 288

Hogan, B., R. Beddington, F. Costantiti and E. Lacy. 1994. Manipulating the Mouse Emryo, 2 nd ed., pp. 129-143, pp. 416. Cold Spring Harbor Laboratory Press. NY, USA. Quinn, P., C. Barros and D. G. Whittingham. 1982. Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J. Reprod. Fert. 66:161-168. Tang, P. C., W. A. Ritchie. I. Wilmut and J. D. West. 2000. The effects of cell size and ploidy on cell allocation in mouse chimaeric blastocysts. Zygote 8:33-43. Tang, P. C. and J. D. West. 2000. The effects of embryo stage and cell number on the composition of mouse aggregation chimaeras. Zygote 8:235-243. Tang, P. C. and J. D. West. 2001. Size regulation does not cause the composition of mouse chimaeras to become unbalanced. Int. J. Dev. Biol. 45:583-590. 289

ph F1 F1 hybrid outbred stock inbred strain ICR 2-cell block pyruvate lactate bovine serum albumin, BSA polyvinylpyrrolidone BSA 4 5 pronucleus hatching Milli-Q 100 mm EDTA 4 37 5% CO 2 95% 100% 5% O 2 5% CO 2 90% N 2 15 290

0.22 µm polyethersulfone, PES0.45 µm cellulose nitrate, CN MWM M16 KSOM CZB PBI M2 100 ml 100 ml mouth pipette 30G, 1/2 5 cc 1 ml 2 ml sterilize pipette 35 mm 115 ml filter unit : 0.22 m PSE 0.45 m CN 5% O 2 5% CO 2 90% N 2 500 ml 200 ml 37 1. 100 ml Milli-Q H 2 O 2. BSA 3. 15 4. BSA 291

5. BSA 6. 1 ml pipette 5% O 2 5% CO 2 90% N 2 7. 4 2 8. ph 7.4 270 290 mosm 1. plug 10 12 18 24 2. 200 µl hyaluronidase, 300 µg/ml in MWM2 5 cumulus cells MWM 3 6 MWM 20 µl MWM 3. 30G 1/2 MWM 5 ml infundibulum MWM MWM 4. morula 16 32 blastocyst hatching 4 5 292

2-cell stage embryo 4-cell stage embryo 8-cell stage embryo morula blastocyst hatching 293

Ali, J., W. K. Whitten and J. N. Shelton. 1993. Effect of culture systems on mouse early embryo development. Hum. Reprod. 93:1110-1114. Lawitts, J. A. and J. D. Biggers. 1991. Optimization of mouse embryo culture media using simplex methods. J. Reprod. Fertil. 91:543-556. Joyner, A. L. 2000. Gene targeting: a paractical approach, 2 nd ed. Oxford University Press, USA. Liu, J., A. Rybouchkin, J. Van der Elst and M. Dhont. 2002. Fertilization of mouse oocytes from in vitro-matured preantral follicles using classical in vitro fertilization or intracytoplasmic sperm injection. Biol. Reprod. 67575-67579. Nagy, A., M. Gertsenstein, K. Vintersten and R. Behringer. 2003. Manipulating the mouse embryo: A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press, USA. Tymms, M. J. and I. Kola. 2001. Gene Knockout Protocols. Humana Press, USA. 294

Component (mg/100 ml) MWM M16 CZB KSOM NaCl 640.0 553.3 476.0 559.5 KCl 35.6 35.6 36.0 18.5 CaCl 2 2H 2 O 25.2 25.0 25.0 KH 2 PO 4 16.2 16.2 16.0 4.75 MgSO 4 7H 2 O 29.4 29.3 29.0 4.95 NaHCO 3 190.0 210.1 211.0 210 Glucose 100.0 100.0 100 3.6 Sodium lactate 261.0 or 434.9 mg of 60% 530 µl (60% syrup) 174 µl (60% syrup) syrup Calcium lactate 46.0 Sodium pyruvate 2.5 3.6 3.0 2.2 Glutamine 15.0 14.5 Streptomycin sulfate 5.0 5.0 7.0 5.0 Penicillin G potassium salt 7.5 6.0 5.0 6.3 Phenol Red 100 µl 1.0 50 L 0.1 2-Mercaptoethanol, 20 mm EDTA-2Na (1% solution) 50 µl 50 µl (100 mm) 4.0 0.38 Ess. Amino acid (50X) 1000 µl Non-ess. Amino acid (100X) 500 µl BSA 300.0 400.0 500.0 100.0 295

Component (mg/100 ml) PBI M2 NaCl 800.0 553.3 KCl 20.0 35.6 CaCl 2 12.0 CaCl 2 2H 2 O 25.2 KH 2 PO 4 20.0 16.2 MgCl 2 6H 2 O 10.0 MgSO 4 7H 2 O 29.3 Na 2 HPO 4 115.0 NaHCO 3 34.9 HEPES 496.9 Glucose 100.0 100.0 Sodium pyruvate 3.6 3.6 Sodium lactate 261.0 or 434.9 mg of 60% syrup Penicillin G potassium salt 7.5 6.0 Streptomycin sulfate 5.0 Phenol Red 1.0 BSA 300.0 400.0 296

DNA DNA DNA DNA DNA DNA DNA DNA DNA chromosome instability CsCl density gradient DNA LB 1% peptone; 0.5% yeast extract; 25 mm NaCl Solution I 50 mm glucose; 25 mm Tris-Cl, ph 8.0; 10 mm EDTA; 5 mg/ml lysozyme Solution II 0.2 N NaOH, 1% SDS, ph 12.5 Solution III 3 M KOAc, 5 M acetic acid, ph 4.8 low-melting point agarose gel; LMP gel 0.5 X TAE buffer Tris-base; acetate; and EDTA Tris-HCl 1 mm; EDTA 0.1 mm; ph 8.0 TE buffer Tris-HCl 10 mm; EDTA 1 mm; ph 8.0 COSMO model Hitach; CS150GX S100NT Neo Vertical Rotor heat sealer dialysis cassette UV 297

DNA DNA RNA DNA DNA DNA DNA DNA DNA DNA DNA 1. 37 5 ml 2. 5 ml 500 ml 15 20 3. 4 6,000 rpm 15 1 4. 5. 10 ml Solution I 5 6. 20 ml Solution II 10 7. 15 ml Solution III 10 8. 4, 12,000 rpm 15 298

9. 2 99.5% EtOH 0.1 3 M NaOAc -20 30 DNA 10. 4 10 13,000 rpm 11. 70% EtOH DNA 2 12. DNA 13. DNA 11 ml TE buffer 14. 11 g CsCl CsCl 15. 0.88 ml EtBr 10 16. 25 3,000 rpm 10 17. 18. 18 13.5 µl Triton-100 19. 20. 100,000 rpm 5.5 21. 18 DNA 22. buffer-saturated butanol DNA EtBr 23. DNA dialysis cassette 16 DNA 1. DNA 2. DNA 3. 0.8% 1.5% LMP 0.5 g/ml EtBr 4. DNA LMP 5. LMP gel 6. 65 10 LMP 7. 0.1 5 M NaCl 5 8. phenol 299

9. 10,000 rpm 10 10. phenol 11. chloroform DNA 12. 2 99.5% EtOH 0.1 3 M NaOAc -20 30 DNA 13. 4 13,000 rpm 10 14. 70% EtOH DNA 2 15. DNA 16. DNA TE buffer DNA 1. DNA 3 ml 3 5 g DNA 2. 3g CsCl CsCl 1 g CsCl/mL 3. 24 L EtBr 10 4. 3,000 rpm 10 5. 18 3.5 L TritonX-100 6. 7. 100,000 rpm 6 8. 18 DNA 9. buffer-saturated butanol DNA EtBr 10. dialysis cassette 16 buffer 11. 300

(A) DNA (B) DNA DNA (A) 1st banding (B) 2nd banding O.C. C.C.C. L. form transgene RNA DNA (A) DNA O.C. open circular form C.C.C. closed covalent circular form DNA (B) DNA L. form DNA 301

Chen, C. M., K. B. Choo and W. T. K. Cheng. 1995. Frequent deletions and sequence aberrations at the transgene junctions of transgenic mice carrying the papillomavirus regulatory and the SV40 TAg gene sequences. Transgenic Res. 4:52-59. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2 nd ed., pp. 9.14-9.19. Cold Spring Harbor Laboratory Press. New York, USA. 302

DNA stereomicroscope pasteur pipette mouth pipette puller, Narishige PN-3, Japan; Sutter P-97, USA microforge, Narishige MF-9, Japan glass capillary tubing 1. holding pipette filament 10 15 cm puller, Narishige PN-3, Japan microforge, Narishige MF-9, Japan 70 100 µm 10 15 µm Sutter Instrument Co., Model P-97 303

2. injection pipette internal glass filament 6150, A-M system, USA DNA -Narishige PN-3 Sutter P-97 1 µm DNA DNA pasteur pipette DNA DNA 3. embryo transfer pipette 120 180 µm burn polish mouth pipette DNA M2 10 20 304

120 180 2 3 3 2 1 M2 M2 ( Hogan et al., 1994 1999) 305

1995 24:181-189 1999 pp. 45-60 Hogan, B., R. Beddington, F. Costantiti and E. Lacy. 1994. Manipulating the Mouse Emryo, 2 nd ed., pp. 173-175, 232-235. Cold Spring Harbor Laboratory Press, NY, USA. 306

recipient mother pseudopregnancy 2.5% avertin 3% pentobarbital 1 ml 27G 75% size 5-0 size 10 10 g 2,2,2-tribromoethyl alcohol Aldrich T4,840-2 10 ml tert-amyl alcohol Aldrich 24,048-6 100% avertin 2.5% 100% 2.5% avertin 4 pentobarbital 30 mg 1 ml g 0.014 0.018 ml 307

2.5% avertin 3% pentobarbital 0.001 ml/g a 1 ml 27G 3 4 2,2,2-tribromoethanol 1. autoclaved 2. a 3. 75% b 4. 1.5 cm b 5. fat pad, c 6. d, e 7. 8. 9. f 10. 308

a b c 309

a b c d e f (a) b 1.5cm c d e f 310

Hogan, B., R. Beddington, F. Costantiti and E. Lacy. 1994. Manipulating the Mouse Emryo. 2 nd ed., pp. 133, pp. 170-172, pp. 416. Cold Spring Harbor Laboratory Press. NY, USA. Pinkert, C. A. 1994. Transgenic animal technology. pp. 80-81. Academic Press, USA. 311

gene transfer transgenic animal gene microinjection embryonic stem cells, ES cells sperm vector retroviral vector infection somatic cell nuclear transfer rearrangement deletion duplication translocation mouserat kb DNA A inverted microscope DIC differential interference contrast Hoffman micromanipulator holding pipette and injection needle micrometer syringe injection chamber B Pasteur pipette M2 medium M16 medium light mineral oil hyaluronidase incubator, 5% CO 2 312

embryo donor recipient vasectomized male petri dish 1. Petri dish 10 mm 35 mm petri dish 3 5 µl petri dish Hoffman 2. 2 4 mm DIC 3. DIC DNA back loading DNA holder 10 20 DNA 37 5% CO 2 313

314

Hoffman (A) (B) (A) Hoffman (microsyringe ) (B)petri dish 35 mm petri dish 315

(A) (B) (A) Hoffman (B) (A) (B) (A) DIC ( ) (B) ( ) 316

Gordon, J. W. 1993. Production of transgenic mice. Methods Enzymol. 225:747-770. Gordon, J. W. and F. H. Ruddle. 1983. Gene transfer into mouse embryos: Production of transgenic mice by pronuclear injection. Methods Enzymol. 101:411-432. Hogan, B., R. Beddington, F. Costantini and E. Lacy. 1994. Manipulating the Mouse Embryo. A Laboratory Manual, 2 nd ed., pp. 237-248. Cold Spring Harbor Laboratory Press. NY, USA. Ju, J. C., S. P. Cheng, P. C. Tarng and K. B. Choo. 1991. In vivo development and micro-injection of rabbit zygotes. Asian. J. Anim. Sci. 4:73-78. Peterson, K. R., C. H. Clegg, C. Huxley, B. M. Josephson, H. S. Haugen, T. Furukawa and G. Stamatoyannopoulos. 1993. Transgenic mice containing a 248-kb yeast artificial chromosome carrying the human β-globin locus display proper developmental control of human globin genes. Proc. Natl. Acad. Sci. 90:7593-7597. 317

vaginal plug oviduct transfer uterine transfer 5 7 morula 20 30% 75% 10 15 19 21 1. 1 0.5 recipient vasectomized 2 Pasteur pipette M2 medium light mineral oil ---2.5% Avertin 1 ml 26G 1/2-inch 90 mm petri dish 70% 318

4-0 2. 1 outbred ICR 2 3 g 0.015 0.017 ml 2.5% Avertin 90 mm petri dish 70% A 4 2 3 cm 1 cm B C 5 bursa D 6 M2 120 180 µm Pasteur pipette M2 medium M2 medium 10 15 A 7 B M2 medium C 8 319

9 4 0 1. 1 2.5 3.5 2 2. 1 4 5 utero-tubal junction 5 mm 26G 6 M2 medium 200 µm medium medium 5 8 7 8 9 320

(A) 70% (B) 2-3 cm 1 cm (C) (D) bursa M2 medium (A) (B) (C) (A) 120-180 µm M2 medium M2 medium 10-15 (B) (C) 321

Utero-tubal junction (A) (B) (A) 26G (B) 3.5 322

Hogan, B., R. Beddington, F. Costantini and E. Lacy. 1994. Manipulating the Mouse Embryo. A Laboratory Manual, 2 nd ed., pp. 237-248. Cold Spring Harbor Laboratory Press. NY, USA. Pinkert, C. A. 1994. Transgenic animal technology. A Laboratory Handbook, pp. 55-57. Academic Press, Alabama, USA. Quinn, P., C. Barros and D. G. Whittingham. 1982. Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J. Reprod. Fert. 66:161-168. Tang, P. C., W. A. Ritchie, I. Wilmut and J. D. West. 2000. The effects of cell size and ploidy on cell allocation in mouse chimaeric blastocysts. Zygote 8:33-43. Tang, P. C. and J. D. West. 2001. Size regulation does not cause the composition of mouse chimaeras to become unbalanced. Int. J. Dev. Biol. 45:583-590 323

DNA DNA genomic DNA polymerase chain reaction, PCR Southern blot hybridization DNA DNA DNA DNA DNA 1. 10 ml 1.5 ml UV 2. 0.9% NaCl Solution A 75 mm NaCl; 255 mm EDTA, ph 8.0 Solution B 10 mm Tris, ph 8.0; 1% SDS Proteinase K solution 10 mg/ml Phenol Chloroform Isoamyl alcohol Isopropanol 5M NaCl 324

70% EtOH TE buffer 5 mm Tris-HCl, ph8.0; 0.1 mm EDTA 0.8% agarose gel EtBr 10 mg/ml 1. 0.5 g 10 ml 2 ml 0.9% NaCl 2. 1500 rpm 10 3. 1 ml Solution A 1 ml Solution B ml Solution B 40 µl proteinase K solution 4. 42 12 5. phenol 6. 3000 rpm 10 10 ml chloroform 1/25 isoamyl alcohol 7. 3000 rpm 10 10 ml 8. 5-7 9. isopropanol 1/10 5M NaCl 10. DNA 1.5 ml 70% EtOH 11. DNA TE buffer DNA 0.8% agarose gel EtBr solution UV DNA DNA TE buffer UV spectrophotometer 260 nm 280 nm DNA A 260 /A 280 1.75 2.0 DNA 260 nm 1 50 µg/ml 325

DNA 1. 10 ml 1.5 ml UV 2. 0.144 M NH 4 Cl 0.9% NaCl TNE buffer 10 mm Tris-HCl; 150 mm NaCl; 10 mm EDTA Proteinase K solution 10 mg/ml Phenol Chloroform Isoamyl alcohol Isopropanol 5M NaCl 70% EtOH TE buffer 5 mm Tris-HCl, ph 8.0; 0.1 mm EDTA 0.8% agarose gel EtBr 10 mg/ml 1. 10 ml 10 ml 2. 3000 rpm 10 90% 3. 7 ml 0.144 M NH 4 Cl 10 4. 3000 rpm 10 0.9% NaCl 5. 2 ml TNE buffer 6. 30 µl proteinase K solution 7. 42 C 12 8. phenol 326

9. 3,000 rpm 10 10 ml chloroform 1/25 isoamyl alcohol 10. 3,000 rpm 10 10 ml 11. 8-10 12. isopropanol 1/10 5M NaCl 13. DNA 1.5 ml 70% EtOH 14. DNA TE buffer DNA 0.8% agarose gel EtBr solution UV DNA 327

Hogan, B., R. Beddington, F. Costantini and E. Lacy. 1994. Manipulating the Mouse Embryo: A Laboratory Manual. 2 nd ed., Cold Spring Harbor Laboratory Press, NY, USA. Horng, Y. M. and M. C. Huang. 2003. Male-specific DNA sequences in pigs. Theriogenology 59:841-848. Sanbrook, J. and D. W. Russell. 2001. Molecular Cloning: A Laboratory Manual. 3 rd ed., Cold Spring Harbor Laboratory Press, NY, USA. Yen, N. T., M. C. Huang and C. Tai. 2001. Genetic variation of RAPD polymorphisms in Taoyuan and Duroc pigs. J. Anim. Breed. Genet. 118:111-118. 328

---Southern blot hybridization Southern blot hybridization DNA genomic DNA DNA restriction enzyme agarose gel DNA DNA probe DNA blocking DNA copy number TAE buffer: 40 mm Tris-acetate ph 8.0, 2 mm EDTA Denaturation solution: 0.5N NaOH, 1.5M NaCl Neutralization solution: 1.0 M Tris-HCl, ph 7.4; 1.5 M NaCl Neutral transfer buffer: 10X SSC Nylon membrane 0.45 µm Schleicher and Schuell Wash solution I: 2X SSC, 0.1% SDS Wash solution II: 0.5X SSC, 0.1% SDS Washing buffer: 100 mm Maleic acid, 150 mm NaCl; ph 7.0; 0.3% Tween 20 Blocking solution: 100 mm Maleic acid, 150 mm NaCl, ph 7.0; 1% w/v blocking reagent Roche Anti DIG antibody, alkaline phosphatase conjugated Roche Detection buffer: 100 mm Tris-HCl, 100 mm NaCl, ph 9.5 CSPD Prehybridization solution: 50% formamide, 6X SSPE, 5X Denhardt s reagent, 0.5% SDS, 100 µg/ml salmon sperm DNA, 1 µg/ml poly (A) 329

hybridization oven agarose gel 1. DNA genomic DNA1 µg/µl 10 µg DNA restriction enzyme 10X 37 1 2 2. TAE 1% w/v 60 65 3. 1X TAE DNA 1 µl loading buffer 125 V 1. 250 mm HCl 10 denaturation solution 15 2 neutralization solution 15 2 2. capillary method 3MM 20X SSC nylon membrane3mm 500 g 16 24 DNA electrophoretic transfer vacuum transfer 1 2 3. UV-crosslinking DNA immobilization UV 120 mj/cm 2 4. 4 6 330

1. Alkali-stable Digoxiginin-11-UTP Roche dttp polymerase chain reaction, PCR -20 2. DNA prehybridization solution DIG Easy Hyb Roche 42 1 3. 10 20 16 4. DNA wash solution I 2X SSC, 0.1% SDS 15 wash solution II 0.5X SSC, 0.1% SDS 64 15 washing buffer 2 5. blocking solution blocking 30 60 6. 1 10,000 DIG alkaline phosphatase Anti DIG antibody, alkaline phosphatase conjugated; Roche 30 washing buffer 15 7. detection buffer 2 8. CSPD, 1 100 in detection solution 5 37 10 15 9. X Biomax, Kodak 1. nick translated random primed 32 P/ 33 P 331

2. DNA prehybridization solution 100 µl/cm 2 42 1 2 3. 5 0.1 1 N NaOH 37 5 denature 4. 2. prehybridization solution 5 25 ng/ml 1-5 10 6 cpm/ml prehybridization solution 10 ml/100 cm 2 42 12 20 5. 7X SSPE/0.5% SDS 15 1X SSPE/0.5% SDS 37 15 0.1X SSPE/1% SDS 65 30 60 6. 3MM X intensifying screen-70 332

3MM 3MM 333

Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Struhl. 1995. Prearartion and analysis of DNA. In: Current Protocols in Molecular Biology, pp. 2.1-2.14. John Wiley and Sons, Inc. NJ, USA. Beck, S. and H. Koster. 1990. Application of dioxetane chemiluminescent probes to molecular biology. Anal. Chem. 62:2258-2270. Chomczynski, P. 1992. One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem. 201:134-139. Sambrook, J. and D. W. Russell. 2001. Preparation and analysis of eukaryotic genomic DNA. In: Molecular Cloning, 3 rd ed., pp. 6.1-6.64. Cold Spring Harbor Laboratory Press, NY, USA. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 334

PCR Slot blot DNA DNA polymerase chain reaction, PCR Slot-blot prehybridization solution 50% formamide, 5X SSC, 50 mm Na 2 HPO 4 ph 6.8, 0.1% SDS, 5X Denhardt s solution, 200 µg/ml denatured salmon sperm DNA hybridization solution 50% formamide, 5X SSC, 25 mm Na 2 HPO 4 ph 6.8, 0.1% SDS, 2X Denhardt s, 5% dextran sulfate, 100 µg/ml denatured salmon sperm DNA, >2 10 6 cpm α 32 P-dCTP nitrocellulose membrane; NC membrane X-ray PCR machine; Perkin Elimer 2400 Slot-blot Minifold II Schleichef and Schuell, Germany UV crosslinker straralinker TM1800; Stratagene, νa hybridization incubator PCR 335

1.5 cm 1.5 ml lysis buffer 100 mm NaCl, 50 mm Tris, 100 mm Na-EDTA, 1% SDS, ph 8.0 proteinase K; 100 µg/ml 55 12 phenol chloroform 12,000 rpm 10 DNA TE DNA DNA DNA 100 ng PCR DNA PCR 3-5 - PCR PCR 50 µl 1X PCR 2.5 mm MgCl 2 200 µm dntp 200 nm 1U Taq polymerase 100 ng DNA PCR Genomic DNA (100 ng) 1.0 µl 10X PCR buffer 5.0 µl 2.5 mm dntp 4.0 µl Primer (upstream): 10 pmol/µl 0.5 µl Primer (downstream): 10 pmol/µl 0.5 µl Pro Taq DNA Polymerase (2 U/µL) 0.5 µl ddh 2 O 38.5 µl 50.0 µl PCR 0.2-mL PCR PCR 35 336

94 1-3 min DNA 94 30 sec 54-60 30 sec 72 PCR 72 1 min 5 min 4 PCR 10 µl 1 µl 1.5% PCR B Slot-blot 5 µg DNA0.3 M NaOH 68 30 l M NH 4 OAc, ph 4.8 DNA Slot-blot Minifold II DNA UV crosslinker 1,200 DNA NC 25 ml 42 2 4 3/4 32 P >2 10 6 cpm cdna 42 16 24 NC 0.1% SDS/2X SSC 15 0.1% SDS/0.1X SSC 48 15 50 15 0.1% SDS/0.1X SSC 52 15 55 15 NC PE X 337

autoradiography Slot-blot DNA 338

PCR (A) PCR (B) PCR NC DNA PCR Slot-blot (A) DNA (B) Slot-blot 339

Chen, C. M., K. B. Choo and W. T. K. Cheng. 1995. Frequent deletions and sequence aberrations at the transgene junctions of transgenic mice carrying the papillomavirus regulatory and the SV40 TAg gene sequences. Transgenic Res. 4:52-59. Chen, C. M., C. H. Wang, S. C. Wu, C. C. Lin and W. T. K. Cheng. 2002. Temporal and spatial expression of biologically active human factor VIII in the milk of transgenic mice driven by bovine -lactalbumin promoter. Transgenic Res. 11:257-268. Chen, C. M., C. L. Hu, C. H. Wang, C. M. Hung, H. K. Wu, K. B. Choo and W. T. K. Cheng. 1999. Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene. Mol. Reprod. Dev. 54:209-214. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, USA. 340

RNA 10-5 µg RNA RNA polymerase transcription RNA messenger RNA, mrna mrna RNA total RNA 1 5%80 85% RNA ribosomal RNA rrna 28S 18S 5S RNA RNA transfer RNA trna 10 15% RNA small nuclear RNA [snrna] small cytoplasmic RNA [scrna] translationmrna splicing mrna RNA DNA RNA DNA CsCl RNA RNase RNA RNase RNA RNase RNase-free ddh 2 O RNase RNase 250 C 4 37 C 0.1% diethylpyrocarbonate DEPC 12 RNase DEPC 100 C 15 RNA RNase RNA 341

0.1% DEPC 12 15 DEPC Tris Tris RNase RNase RNase RNA -80 C RNA RNA RNase RNA RNase inhibitor DEPC vanadyl-ribonucleoside complex RNasin Guanidinium isothiocyanate guaninidium chloride RNase RNA RNase PCR RNA RNA homogenizer Eppendorf 1. ddh 2 O/Diethylpyrocarbonate DEPC 0.1 ml DEPC 100 ml ddh 2 O 4 DEPC 121 C 45 DEPC 2. Denaturing solution 342

Stock solution ddh 2 O/DEPC 293 ml 0.75 M sodium citrate ph 7.0 17.6 ml 10% w/v N-lauroylsarcosine Sarkosyl 26.4 ml guanidine thiocyanate 250 g 60 65 C Working solution 0.35 ml β-mecaptoethanol 50 ml stock solution 4 M guanidine thiocyanate, 25 mm sodium citrate, 0.5% Sarkosyl, and 0.1 M β-mecaptoethanol 3. 2M Sodium acetate 16.42 g sodium acetate anhydrous 40 ml 35 ml glacial acetic acid glacial acetic acid ph 4 100 ml 4. Water-saturated phenol 60 65 C 100 g phenol crystals 4 C 5. 49:1 v/v chloroform/isoamyl alcohol bromochloropropane 6. 100% isopropanol 7. 75% ethanol DEPC ddh 2 O 8. deionized formamide 1. 100 mg 5 10 10 6 1 ml denaturing solution pipetman 2. 0.1 ml 2 M sodium acetate 1 ml Water-saturated phenol 0.2 ml 49:1 v/v chloroform/isoamyl alcohol or bromochlo- ropropane 15 3. 4 C 10,000 g 20 RNA aqueous phase 1 ml DNA organic phase 4. 1 ml 343

1 ml isopropanol -20 C 30 5. 4 C 10,000 g 10 pipetman 6. 0.3 ml isopropanol -20 C 30 4 C 10,000 g 10 pipetman 7. 1 ml DEPC 75% 10 15 8. 4 C 10,000 g 5 5 10 RNA 9. RNA ddh 2 O/DEPC formamide 55 60 C 10 15 RNA 10. -80 C TRIzol RNAzol TM B RNA Kit RNA Kit RNA TRIzol GibcoBRL/Invitrogen, Life Technologies RNAzol TM B Tel-Test Inc. homogenizer Eppendorf Chloroform Isopropanol 75% Ethanol DEPC ddh 2 O TRIzol 1. 50 mg 5 10 10 6 1 ml TRIzol 344

pipetman 2. 0.2 ml chloroform 15 vortex 5 3. 4 C 12,000 g 15 phenol-chloroform organic phase RNA aqueous phase 60% DNA 4. 0.5 ml 0.5 ml isopropanol 10 5. 4 C 12,000 g 10 pipetman 6. 1 ml ddh 2 O/DEPC 75% 4 C 7,500 g 5 5 10 RNA 7. RNA ddh 2 O/DEPC 0.5% SDS 1 mm EDTA ph 7.0 55 60 C 10 15 RNA 8. -80 C RNAzol TM B 1. 50 mg 5 10 10 6 1 ml RNAzol TM B pipetman 2. 0.2 ml chloroform 15 vortex 15 3. 4 C 12,000 g 15 phenol-chloroform organic phase RNA aqueous phase 50% DNA 4. 0.5 ml 345

0.5 ml isopropanol 15 5. 4 C 12,000 g 10 pipetman 6. 1 ml ddh 2 O/DEPC 75% 4 C 7,500 g 8 5 10 RNA 7. RNA ddh 2 O/DEPC 0.5% SDS 1 mm EDTA ph 7.0 55 60 C 10 15 RNA 8. -80 C RNA RNA ddh 2 O/DEPC UV spectrophotometer 260 nm 280 nm RNA A 260 /A 280 1.7 2.0 DNA 1.8 RNA 260 nm 1 40 µg/ml DEPC Phenol Chloroform Isoprpanol TRIzol RNAzol TM B 4 C 12,000 g 10 RNA RNA RNA storage solution (Ambion) 4-70 346

2001 - pp. 43-66 Chomczynski, P. and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159. Gilman, M. 2001. Section I preparation of RNA from eukaryotic and prokaryotic cells-preparation and analysis of RNA. In: Current Protocols in Molecular Biology. John Wiley and Sons, Inc. NJ, USA. Sambrook, J. and D.W. Russell. 2001. Extraction, purification, and analysis of mrna from eukaryotic cells. In: Molecular Cloning, A Laboratory Manual. 3 rd ed., pp. 7.1-7.88. Cold Spring Harbor Laboratory Press, NY, USA. 347

---Northern blot hybridization & RT-PCR Northern blotting hybridization Northern blot hybridization mrna RNA denaturing agarose gel electrophoresis capillary transfer RNA nylon membrane mrna RNA RNA polynucleotides ribonucleoprotein particles RNPs conformation RNA RNA denature RNA RNA cdna mrna cdna reverse transcriptase cdna DNA I T4 DNA cdna cdna 3 - Sl cdna cdna cdna cdna cdna library glyoxal/dimethyl sulfoxide DMSO formaldehyde Glyoxal/DMSO ph 348

RNA RNA formamide RNase RNA RNA RNase RNA DNA RNA 3% H 2 O 2 10 rrna RNA 80 85% ethidium bromide EtBr RNA large small rrna 28S 18S 5S RNA trna 18S rrna smear mrnas mrna UV transilluminator 3% H 2 O 2 ddh 2 O/DEPC RNA 12.3 M 37% Formaldehyde, ph>4.0 10X Formaldehyde gel-running buffer: 0.4 M MOPS, ph 7.0 10X MOPS running buffer: 0.1 M Sodium acetate; 0.01 M EDTA, ph 8.0 Formaldehyde gel-loading buffer: 1 mm EDTA; 0.25% Bromophenol blue; 0.25% Xylene cyanol; 50% Glycerol Ethidium bromide EtBr, 1 mg/ml in ddh 2 O/DEPC Agarose *MOPS 3-(N-morpholino)-propanesulfonic acid 349

1. 2. 3% H 2 O 2 10 H 2 O 2 3. ddh 2 O/DEPC 4. 1.0% 1 g agarose 250 ml 72 ml ddh 2 O/DEPC 5. 60 C 5 30 6. 10 ml 10X Formaldehyde gel-running buffer 18 ml (teeth comb) 30 7. 1.5 ml Reagent µl ddh 2 O/DEPC + RNA (0.5-10 µg) 11 10X Formaldehyde running buffer 5 12.3 M Formaldehyde 9 Formamide 25 Total volume 50 8. 65 C 15 9. RNA 10 µl formaldehyde gel-loading buffer 2 µl EtBr EtBr ddh 2 O/DEPC 10. 1 formaldehyde gel-running buffer 5 V/cm 5 11. RNA 5 V/cm bromophenol blue BPB 2/3 UV transilluminator box RNA 350

RNA nitrocellulose membrane nylon membrane blotting electroblotting RNA 6 RNA polyacrylamide RNA RNA UV irradiation 80 C 2 Nylon membrane Whatman 3MM 20X SSC 0.3 M Sodium citrate, ph 7.0 3 M NaCl ddh 2 O/DEPC 1. ddh 2 O/DEPC 2. 100 ml 20X SSC 5 3. RNA 4. 1 20X SSC 2 351

3 DNA/RNA probe DNA/RNA 32 P 35 S autoradiography 32 P 35 S digoxigenin, DIG biotin DNA nick translation random priming polymerase chain reaction PCR DNA DNA [α- 32 P] datp [α- 32 P] dctp DIG-11-dUTP DNA end-labeling filling-in polynucleotide kinase reaction DNA random priming PCR Random priming DNA DNA primer 3'-OH extension DNA de novo synthesis random primers DNADNA random priming Klenow fragment large fragment of E. coli DNA polymerase I DNA [α- 32 P]dNTP DIG-11-dUTP random priming DNA template DNA random priming 1. 37 C 352

2. DNA 0.2 M EDTA, ph 8.0 4 M LiCl Random priming DIG Kit Boehringer Mannheim; Roche 100% ethanol TE buffer 10 mm Tris-HCl, ph 8.0; 1 mm EDTA 3. 1 100 ng DNAddH 2 O 15 µl 2 10 3 2 µl hexanucleotide mixture + 2 µl dntp mixture + 1 µl Klenow enzyme 37 C 2 42 µl 0.2 M EDTA DNA 5 2.5 µl 4 M LiCl 75 µl 6 DNA 50 µl TE buffer Polymerase chain reaction PCR PCR DNA PCR DNA multiple cloning sites DNA SP6 T3 T7 promoter primer M13/pUC forward reverse primers PCR [α- 32 P] dntp DIG-11-dUTP DNA 32 P DIG PCR 1. PCR 2. DNA T3 promoter primer T7 promoter primer 2 mm 353

PCR PCR DIG probe synthesis kit Roche Taq DNA polymerase Expand High Fidelity, 3.5 U/µL ; 10X PCR DIG probe synthesis mix 2 mm datp, dctp, dgtp, 1.9 mm dttp, 0.1 mm DIG-11-dUTP, ph 7.0 Expand high fidelity buffer with MgCl 2 10X 3. PCR DIG probe synthesis kit 1 0.2 ml Reagents Volume (L Final concentration ddh 2 O 28.5-10 PCR buffer 5 1 10 PCR DIG mix 5 200 µm dntp T3 promoter primer 5 0.2 µm T7 promoter primer 5 0.2 µm pblusgus DNA 1 ~50 pg Taq DNA polymerase 0.5 0.5 U Total volume 50 2 3 100 µl PCR 4 PCR PCR Denaturation 94 C/10 extension30 [94 C/45 55 C/1 72 C/2 ] elongation step 72 C/10 5 PCR 6 5 µl PCR 7 DIG- DNA phenol/chloroform DIG organic phase 354

1. probe RNA 2. SDS 1. RNase 2. prehybridization 3. random priming PCR DNA denature 4. stringency 42 C 50 C hybridization oven Whatman 3MM hybridization tube 6X SSC Nylon membrane probe 50X Denhardt s solution 5 g Ficoll + 5 g polyvinylpyrrolidone + 5 g BSA fracitonv, 500 ml 0.45 µm hybridization buffer 6X SSC; 0.5% SDS; 5X Denhardt s solution; Denatured salmon sperm DNA 100 µg/ml 2X SSC 0.1% SDS 0.2X SSC 0.1% SDS 0.1X SSC 0.1% SDS 1. well 2. 20 ml 6X SSC 5 3. Whatman 3MM 30 355

4. Crosslinker UV 5. 10 ml hybridization buffer 42 C prehybridization 1 2 6. 42 C 7. 80 ml 2X SSC 0.1% SDS 5 Geiger counter 8. 100 ml 0.2X SSC 0.1% SDS 42 C 15 9. 100 ml 0.1X SSC 0.1% SDS 50 C 15 10. Whatman 3MM 11. DIG anti-dig 酶 alkaline phosphatase, AP conjugate 酶 酶 substrate NBT nitroblue tetrazolium BCIP 5-bromo-4- chloro-3-indolyl phosphate, toluidinium salt CSPD CPD-star shaker DIG nucleic acid detection Kit Soultion 0.1 M maleic acid; 0.15 M NaCl, ph 7.5 Soultion 1% blocking reagent in Soultion Soultion 0.1 M Tris-HCl, ph 9.5; 0.1 M NaCl; 50 mm MgCl 2 356

Soultion 10 mm Tris-HCl, ph 8.0; 1 mm EDTA Washing soultion: 0.3% Tween 20 in Soultion Color-substrate solution 200 µl NBT/BCIP stock solution plus 10 ml Soultion 1. 50 ml Washing solution 1 2. 20 ml Soultion 30 3. 2 µl anti-dig-ap-ab 10 ml Soultion 2 50 rpm 30 4. 50 ml Soultion 2 15 Ab 5. 20 ml Soultion 2 6. 10 ml color-substrate solution 7. 50 ml Soultion 5 8. 30 Reverse transcriptase-ploymerase chain reaction RT-PCR mrna cdna mrna cdna cdna mrna cdna cdna ph ph 8.3 cdna ±0.5 ph cdna 5 mrna dntp cdna dntp 10 50 µm cdna 90 45 357

RNA ddh 2 O/DEPC 10 mg/ml Bovine serum albumin BSA 5 U/µL RNasin 350 mm β-mecaptoethanol 50 µl 14.4 M β-mecaptoethanol + 1,950 µl ddh 2 O/DEPC 10X RT buffer 100 mm Tris-HCl, ph 8.3; 80 mm NaCl; 16 mm MgCl 2 100 mm dntp datp, dttp, dctp, dgtp 200 mm Sodium pyrophospate Primers oligo(dt) 12-18 random hexamer 100 ng/µl ddh 2 O/DEPC Reverse transcriptase 1. 2 µg RNA ddh 2 O/DEPC 18 µl 2. RNA 65 C 5 denature 3. 2.5 µl 10X RT buffer 3 pmol 1 2 µl ddh 2 O/DEPC 25 µl 4. oligo(dt) 4 C 15 random hexamer 15 5. Reagent µl 10 RT buffer 2.5 BSA 5 µg/µl 1 RNasin 5 U/µL 1 350 mm β-mecaptoethanol 2 100 mm datp 0.5 100 mm dttp 0.5 100 mm dctp 0.5 100 mm dgtp 0.5 200 mm Sodium pyrophospate 1 ddh 2 O/DEPC 14.5 Total volume 24 6. RNAs/primers 25 µl reverse transcriptase 1 µl 50 358

7. 42 C 45 cdna single strain complementary DNA 8. -20 C PCR ThermoScript TM RT-PCR System ThermoScript TM RT-PCR System Invitrogen, Life Technologies Reagent µl ThermoScript TM RT 15 U/µL 100 5 cdna synthesis buffer 500 0.1 M Dithiothreitol DTT 250 RNaseOUT TM 40 U/µL 100 10 mm dntp Mix 2 250 Oligo (dt) 20 50 µm 100 Random Hexamers 50 ng/µl 250 E. Coli RNase H 2 U/µL 2 50 DEPC-Treated Water 1.5 ml 1. Reagent µl RNA 10 pg 5 µg X Primer 1 0.1 M DTT 1 10 mm dntp Mix 2 DEPC-Treated Water X Total volume 12 2. 65 C 5 RNA 3. 5 4. 8 µl cdna synthesis mix Reagent µl ThermoScript TM RT 15 U/µL 1 5 cdna synthesis buffer 4 0.1 M DTT 1 RNaseOUT TM 40 U/µL 1 DEPC-Treated Water 1 5. oligo(dt) 20 50 60 C 359

30 60 random hexamer 10 50 60 C 20 50 cdna 6. 85 C 5 7. 1 µl RNase H 37 C 20 RNA 8. -20 C 2 µl PCR 1. DEPC formaldehyde formamide ethidium bromide 2. UV 360

2001 - pp. 43-66 Brown, T. 2001. Section II analysis of RNA structure and synthesis - preparation and analysis of RNA. In: Current Protocols in Molecular Biology. John Wiley & Sons, Inc., Hoboken, NJ, USA. Edwards, J. B. D. M., P. Ravassard, C. Icard-Liepkalns and J. Mallet. 1995. cdna cloning by RT-PCR. In: PCR 2-A Practical Approach. pp. 89-118. IRL Press, NY, USA. Sambrook, J. and D.W. Russell. 2001. Extraction, Purification, and Analysis of mrna from Eukaryotic Cells. In: Molecular Cloning: A Laboratory Manual. 3 rd ed., pp. 7.1-7.88. Cold Spring Harbor Laboratory Press, NY, USA.. 361

protein purification chromatography electrophoresis NaCl Tris Sodium carbonate Bovine serum albumin Microplate HPLC Micrococcus lysodeikticus 5 mla 0.05M NaCl; 50 mm Tris, ph 9.5 10mL 10,000 g 3 362

A CM 100 mm 5 mm 0.5 ml A 5 B 0.05 M NaCl; 0.2 M Sodium carbonate, ph 10.5 fraction 0.5 ml 20 µl 50 µl A Ultrahydrogel 250, Waters, 7.8 mm 300 mm 10 0.8 ml/ 0.5 ml 20 µl 80 µl 10 mg Micrococcus lysodeikticus 3 ml 0.1 M phosphate buffer, ph 7.0 75 ml 20 µl 180 µl microplate 40 30 450 nm O.D. 10 25 ph 7.0 450 nm 0.001 A 450 = 0.001 bovine serum albumin, BSA BCA 1 reagent B 50 reagent A Working reagent WR25 µl BSA microplate 200 µl WR 30 60 30 562 nm O.D. 363

reducing 12% SDS-PAGE 7.mm 8 mm 50 µl sample buffer 50 µl 95 5 5 25 µl 1D Purificational Step Crude CM Size Exclusion Volume (ml) Protein Conc. (mg/ml) Total Protein (mg) Lysozyme (mg) Total Activity % Weight Specific Activity Recovery Fold pure 364

365

2002 : http://140.112.78.220/ Ford, S. R. 2002. Biochemistry 2344 Lecture 10: Protein Purification. http://opbs.okstate.edu/~ford/2344_lab/2344%20lab%20lect ures/lecture%2010--enzyme%20purification/2344%2flectur e%2010-lysozyme%20purif Millard, J. T. 2002. Experiment 3: Purification and characterization of the enzyme lysozyme. http://www.colby.edu/chemistry/ch367/laboratory/experiment 3.pdf Laemmli, U. 1970. Cleavage of structural protein during the assembly of the head of Baxcteriophage T4. Nature 227:680-685. Pierce com. 2002. BCA Proein Assay Kit. http://www.bmb.psu.edu/sbf/biotc479/piercebc.pdf5 366

---Western blot hybridization Western blot hybridization antibody antigen SDS-PAGE nitrocellulose membrane transcription mrna Homogenate buffer: 20 mm Tris-HCl, ph 7.0; 5 mm EDTA; 1 mm PMSF phenylmethylsulfonyl fluoride ; 50 mm 2-mercaptoethanol; 0.25 M sucrose; 0.1% v/v Triton X-100 SDS-PAGE sample buffer 8 ml: 4 ml ddh 2 O; 1 ml 0.5 M Tris ph 6.8 ; 0.8 ml glycerol; 1.6 ml 10% SDS; 0.4 ml 2-mercaptoethanol; 0.2 ml 0.5% bromophenol blue SDS-PAGE electrode buffer 1 L, ph 8.3: 3.0 g Tris; 14.4 g glycine; 5 ml 20% SDS Transfer buffer 1 L: 3.0 g Tris; 14.4 g glycine; 200 ml methanol TBS: 50 mm Tris, ph 7.5; 200 mm NaCl TTBS: 50 mm Tris, ph 7.5; 200 mm NaCl, 0.1% Tween 20 Blocking solution: 5% skim milk TTBS Hybridization solution: 1% skim milk TTBS primary antibody conjugated with horseradish peroxidase; HRP ECL; Amersham Nitrocellulose membrane 0.45 µm; S&S 3MM filter paper 367

SDS-PAGE 1. homogenate buffer 1 10 w/v 4 10,000 g 10 supernatant Bradford 1976 BSA 2. 50-100 µg SDS-PAGE sample buffer 1 1 Laemmli 1970 SDS-PAGE 5% stacking gel 7.5-15% separating gel gradient 3. 100V 200V 35 ma Bromophenol blue 1. Towbin 1979 transfer buffer S&S 3MM 20 kda 0.45 µm 20 kda 0.2 µm 2. 3MM Bio-Rad, USA 3. 4 30 16 60 1 2 hybridization and detection 1. TBS 2 368

5% skim milk TTBS blocking solution 1 blocking Blocking solution 3% gelatin bovine serum albumin 2. blocking 1% TTBS 1 2 3. TTBS 3 5 goat anti mouse IgG horseradish peroxidase conjugated goat anti rabbit IgG horseradish peroxidase conjugated alkaline phosphatase conjugated 1% TTBS 1 4. TTBS 3 5 TBS 5 Tween-20 5. ECL Amersham RPN 2106 alkaline phosphatase conjugated CSPD A B 1 2 X Biomax; Kardo 1 2 369

3MM 3MM 370

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06-3028037 E-mail halnwu@mail.ncku.edu.tw 04-22862799 04-22840265 E-mail jcju@dragon.nchu.edu.tw Dept. of Developmental Biology, University of Cincinnati, USA, Ph.D. Children's Hospital Medical Center, Cincinnati, USA, PDF Assistant research Fellow, IMB, 02-27895547 02-27895588 E-mail htwu@sun5.nlac.gov.tw Academia Sinica, Taipei present Associate research Fellow, IMB, Academia Sinica, Taipei 02-27880460 02-27826085 E-mail hungli@ccvax.sinica.edu.tw PURDUE TUFTS 02-27899568 02-27824595 E-mail zojlwu@ccvax.sinica.edu.tw 037-672352 352 037-692806 E-mail khlee@mail.atit.org.tw 037-672352-588 037-692806 E-mail scw01@mail.atit.org.tw 06-2353535 5541 372

06-5911211 205 06-5911210 E-mail snlee@mail.tlri.gov.tw 02-23123456 6907 (92.1.10 02-23817083 ) E-mail swlin@ha.mc.ntu.edu.tw 04-22872071 04-22872071 E-mail sllee@dragon.nchu.edu.tw 04-22857109 04-22860265 E-mail pctang@dragon.nchu.edu.tw 037-672352 360 037-692806 E-mail cftu@mail.atit.org.tw (1995-present) 02-87923100 18180 02-27895502 E-mail simon@ndmctsgh.edu.tw 03-9357400 818 03-9354794 E-mail lincc@ilantech.edu.tw 06-591121 235~237 06-5911210 E-mail lrchen@mail.tlri.gov.tw 03-8565301 7159 Ellis Fischel 03-8571917 E-mail srlin@mail.tcu.edu.tw 373

04-22856309 04-22851797 E-mail chchen1@dragon.nchu.edu.tw The Sanger Institute, Wellcome Trust, Cambridge, U.K. 0920912676 04-22850223 02-86483544 04-22860265 E-mail yangjame@ms26.hinet.net E-mail mchuang@mail.nchu.edu.tw 03-9357400 811 04-8511888 4252 03-9354794 04-8511666 E-mail mcchen@ilantech.edu.tw E-mail kater@mail.dyu.edu.tw 02-28267177 02-28225044 E-mail slfu@ym.edu.tw 02-23123456 6907 02-23817083 E-mail sharon.ch6005@msa.hinet.net 03-8565301 7171 038-578386 E-mail tzeng@mail.tcu.edu.tw 374

06-2758024 E-mail taichein@mail.ncku.edu.tw 06-5911211 234 06-2758024 E-mail jjt@mail.tlri.gov.tw Dept. of Developmental Biology, University of Cincinnati, USA, Ph.D. 04-23590281 113 02-27361661 3229 04-23590385 02-23774207 E-mail Brou@mail.thu.edu.tw E-mail hsiumei@tmu.edu.tw 02-23639291 124 02-23638483 E-mail hjtsai@ccms.ntu.edu.tw 02-27337180 02-27324070 E-mail wtkcheng@ccms.ntu.edu.tw Davis 06-2757575 65620 375

A α-1,3-galactosyltransferase α-gt α- 100,180 2-cell block 63,290 3-dimentional structure 132 Absorption filter 138 Acrosome 24 Activation 26,41 Adenovirus 58,265 Adult stem cells 52 Aequorin 133 Affinity chromatography 251 Agarose gel 325,330 Aggregation 53,137 Alternating R-Y - 196 American Fisheries Society 149 Amino acid 214 Ampholyte 252 Amphotropic 59,64 Animal cloning 34 Antibiotics 155 Antibody 367 Antigen 367 Apoptosis 73,91 Aqueous phase 343,345 Aspiration 177 Atresia 22,35 Atretic follicle 34 Autofluorescence 133 Autoradiography 90,338,352 B Background emission 137 Backcross 225,276 Barrier 268 Barrier facility 270 Barrier filter 139,140 Bioavailablity 266 Biohazard 265,271 Bioreactor 71,79,212,215 Blastocyst injection 52 Blastocyst 155,292 Blastomeres 27,123 Blotting 256,351 Body composition 213 Bovine papilloma virus BPV 7 Bovine serum albumin BSA 257,290,363 Burn polish 304 C Cage card 271 Capillary method 330 Capillary transfer 348 cdna library cdna 348 CFP 134 Chemical enucleation 177 Chiasmata X- 19 Chimera 53,175 Chimeric mice 156 Chimeric protein 133 Chromatic beam splitter CBS 139 Chromatography 362 Chromophore 132 Chromosome instability 297 Coding region 161 Colored filter 138 Colorimetric assays 258 Complementary color 134 Concatamerization 189 Continuous spectrum 138 Core protein or capsid 58 Counterion 252,255 CsCl density gradient 297 Cumulus cells 177,292 Cysteine 214 Cystic fibrosis CF 223,267 Cytostatic factor CSF 37 D Daily weight gain 213 Denaturing agarose gel electrophoresis 348 Diakinesis 19 Dichromatic mirror DM 139 Dictyate 22,36 Diethylpyrocarbonate DEPC 341 Differentiation 51 Digoxigenin DIG 352 Diphtheria DT-A 158 Diplotene 19,35 DNA linker protein DNA 94 DNA/liposome complex DNA 94 Dosage effect 160 Drug resistance gene marker 266 E Ear tag 271 Ecotropic 59,64 Electrical breakdown 9 376

Electroblotting 351 Electrophoresis 362 Electroporation 5,92,144,155,159 Eluent 250 Elution 251,252 Embryo 239 Embryo cloning 183,229 Embryo transfer 270 Embryo transfer pipette 304 Embryonic germ cells EG cells52 Embryonic stem cell line 155 Embryonic stem cells ES cells 52,89,103,137,211,312 Embryonic stem cell-like 177 Embryonic-derivated stem cells 144 Emission wavelength 132 End point 273 Endogenous gene 265 Enhanced GFP EGFP 132 Enhancer 6,59,230 Enhancer sequences 8,196 Enviropig TM 112 Enzymology 255 Episome 93 Equatorial segment 90 Ex vivo culture 119 Excisionase Xis 190 Excitation filter ( ) 139 Excitation wavelength 132 Exogenous gene 265 Exon 161,203 F F1 hybrid F1 276,290 Feeder cells 156 Female pronuclei 27 Fertilization 17,25 Fetal cells 178 Fibroblast 50,51,173 Flow cytometry 134 Fluorescence 131 Folding 132,162 Folliculogenesis 34 Formaldehyde 348 Foster mother 5,155 Founder 277 Frankenfish 150 Fusion protein 133,165 Fusion tag 136,141 G Gametogenesis 21 Gel filtration 249 Gene integration 110 Gene knockout 51,103,155,164,172,219,222 Gene targeting 51,52,103,155,172 Gene therapy 48,189 Gene transfer 144,312 Gene trap 225 Genetic modification organism GMO 65 Genital ridge 17,21 Genome 189,266 Genomic DNA DNA 79,324,329,330 Genotype 267 Germ line transmission 156,165 Germinal vesicle breakdown GVBD 36 Germinal vesicle GV 22,36,108 Germline chimera 122 Germline transmission rate 62 Glass capillary tubing 303 Gnotobiotic 267 Gonadotropins 22,34,228 Gradient 368 Granulosa cells 177 Green fluorescent protein GFP 131,141 Growth hormone 212 Growth hormone-release factor GHRF 212 H Hatching 290,292 Help cells 59 Hemizygote 111 Heterozygous +/- 155 High-efficiency particulate filter 268 High-pressure mercury short arc lamp 132,137 High-pressure xenon short arc lamp 138 Hit and run 162 Holding manupilator 145 Holding pipette 202, 303,312 Homogenizer 342,344 Homologous chromosomes 18 Homologous recombination 51,125,155,190,229,275 Homo-R/-Y 196 Homozygote 268,285 Homozygous -/- 156,276 Host protein 136 Human chorionic gonadotropin HCG 176,286 Human decay accelerating factor hdaf 99,180,216 377

Human factor IX hfix 100 Human immunodeficiency virus HIV 58 Human leucocyte antigen class II HLA-II 180 Human tissue plasminogen activator tpa 1 Human tubal fluid HTF 41 Hyaluronidase 286,292,312 Hybridization 348 Hydrophilic 251 Hyper-acute rejection 216 Leptotene I 18,35 Identification Leukaemia inhibitory factor LIF 271 Illegitimate recombination 191 156 Immunoglobulin 214,257 Ligand 251 In and out Line spectrum 162 137 In vitro Lipofection 133 126 In vitro culture IVC Liposome 34 92,144 In vitro fertilization IVF Long terminal repeat sequences LTR 34,95 In vitro maturation IVM 59 34,38 In vivo Low-melting point agarose gel 131 Inbred strain LMP gel 290 297 Incident Luciferase 137 133 Individual ventilated cage IVC 268 Luciferin 133 Influenza virus Lytic cycle 215 6 Infundibulum 292 M Inhibitor 342 Major histocompatibility complex II Injection manupilator 145 II 91 Injection needle 202,312 Male pronucleus 26,92 Injection pipette 304 Mammary gland factor MGF 206 Inner cell mass ICM 52,62,103,156 Manipulation of oocyte-enclosed preantral follicle Insertion sequences IS 190,195 38 Insertional mutation im 265,276 Mastitis 215 Insertional recombination 189 Matrix 251 Insulin-like growth factor IGF-1 Maturation promotion factor MPF 212 37,177 Integrase Int 58,190 Medaka Oryzias latipes 5,144 Intercross 276 Meiosis 17 Interference filter 138 Messenger RNA mrna RNA 341 Interferon 215 Methylation 64,205 Internal deletion 194 Micelle 213 Internal glass filament 304 Microforge 303 Internal ribosome entry site IRES Microinjection 8,144,155,229 61 Micro-isolator 268 Interphase 20 Micropyl 145 Intrachromosomal recombination Milk protein binding factor MPBF 163 206 Integration 58 Mobile elements 265 Intron 161,203 Monomer 133 Inverted repeats 74,163,193 Morula 292,318 Isoelectric point pi 249,253 378 Isogenic 157 J Jellyfish 131 K Knock in 163 L Lactase 214 Lactate 45,290 Lactoferrin LF 214,248 Lactose 213 Lampbrush chromosome 19

Mosaic 60,62 Mosaicism 95,147 Mouse 101,155,312 Mouth pipette 291,303,304 Multicolor fluorescence 134 Multiple cloning sites MCS 158 Mutant mt 132 N Negative regulator 213 Negative selection marker 158 Neomycin 157 Nitrocellulose membrane 109,335,351,367 Nonhomologous insertion 275 Non-invasive 131,141 Nonpermissive 6 Northern blot hybridization 348 Nuclear transfer 211,217,219 Nuclear transplantation 51 Nylon membrane 330,348,351 Primordial follicle 22 Primordial germ cells PGC 17,34,52,118 Prion 233,235,265,266 Probe 159,329,352,355 Promoter 2,6,59,157,212,231 Pronuclear microinjection 211 Pronucleus 290 Protein purification 362 Provirus 7,60 Pseudopregnancy 307 Puller 303 Pyruvate 45,290 R Random primers 352 RCAS-TVA RCAS -TVA 71 Real time 131,141 Rearrangement 65,91,110,113,165,312 Recapture 271 Recipient mother 307 O Recombinase 161,163 Oogenesis 21 Recrystallization 243 Organelles 244 Refinement 267,272 Organic phase 343,345 Regulator sequence 203 Outbred stock 276,290 Replication-defective 58 Overlapping 194 Reporter gene 131,159,226 Repressor 165 P Reprogram 102,219 Packaging cells 59 Restriction enzyme 157,329,330 Particle bombardment 144 Resumption of meiosis 36 Pasteur pipette 286,303,304,312,318 Retroviral vector infection 312 Perivitelline space 91 Retrovirus 7,58,265 Phenotype 156,161,267,272 Reverse transcriptase 58,348,358 Phosphorescence 131 Reverse transcriptase-ploymerase chain Reaction Photogenic cell 133 RT-PCR 357 Plasmid 7,164 RFP 134 Plasticity 52 Ribosome RNA rrna RNA 341 Plug 292 RNA interference RNA 71,72 Pluripotency 52,156 RNA splicing RNA 7 Polar body 18,20 Polymerase chain reaction PCR S 92,324,331,335 Salmo salar 150 Positive selection marker 158 SDS-Polyacrylamide gel electrophoresis SDS-PAGE Post-acrosomal region 90 SDS- 255 Potassium simplex optimized medium KSOM Secondary follicle 22,35 45 Sentinel animal 269 Precipitation 248 Separating gel 368 Pregnant mare s serum gonadotropin PMSG Site-directed mutagenesis 162 286 Site-specific recombination 190 Prehybridization solution 332,335 Skim milk 367,369 Primary antibody 367 Southern blot hybridization Primary follicle 22,34,35 5,54,92,125,147,159,193,203,226,324,329 Primer 159,352 Spacer arm 251 379

Specific pathogen free SPF 268 Sperm vector 211,312 Spermiogenesis 23 Sperm-mediated gene transfer 144 Splicing 79,161,203,341 Squeeze 177 Stacking gel 368 Standard operation procedure SOP 269,273 Stereomicroscope 303 Stop codon 162 Stringency 355 Structural gene 203 Substrate 356 Subzona perivitelline space SPS 60 Supernatant 368 Synapsis 18 Syngamy 27,61 380 Transposase 191 Transpositional recombination 190 Trexler-type isolator 268 Two-colored fluorescent mouse 134 U Ultraviolet absorption method UV 257 Unpredictable gene expression or Transgene instability 266 Utero-tubal junction 320 UV irradiation 351 UV spectrophotometer 346 V Vaginal plug 286,318 Vasectomize 318 Vector 6 Viral receptor 265 Viral vector 265 T Vitrification 244 Tag and exchange 163 W Tandem repeat 65,193 Western blot hybridization 110,256,367 Target gene 72,155 Wild-type wt 132,156 Targeting vector 155,157 Telomere 182 X Template 352 Xenotransplantation 216,266 Terminator 144 Xenotropic 59,64 Testis-mediated gene transfer 95 Y Tet-operator tet- 165 YFP 134 Tetracycline tet 165 Tetraploid embryo 4n 137,141 Z Thymidine kinase TK 158 Zebrafish Danio rerio 5,144 Tissue-specific gene expression Zona pellucida ZP 17,22,35,91 1 Zygotene 18,35 Total RNA RNA 341 Totipotency 27,172 Trans-acting factor 189 Transactivation domain 165 Transactivator tta 165 Transcription 158,341,367 Transcription factor 162 Transdominate effect 160 Transfection 122,164,211 Transfer RNA trna RNA 341 Transformation 189 Transgene 265,274 Transgenic animal 45,89,312 Transgenic fish 144 Translation 158,341 Transmissible spongiform encephalopathy 100 Transmittance 138