動物簊因轉殖技術與實驗\(總-送印版\)

Transcription

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2 2008

3 :

4 RNAi RCAS-TVA DNA I

5 DNA Southern blot hybridization PCR Slot blot 335 RNA Northern blot hybridization & RT-PCR Western blot hybridization II

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

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

9 2002β- / 31: : 30:45-48 Houdebine L. M The methods to generate transgenic animals and to control transgene expression. J. Biotechnol. 98: Houdebine L. M., S. Rival, T. Pantano, G. Jolivet, D. Thepot and J. Attal Transgenesis for the study and the control of lactation. Reprod. Nutr. Dev. 42: Huang, M. C. and J. S. Chao Regulatory sequences of the 5' flanking region of goat β-casein gene. AJAS 14: Pintado B. and A. Gutierrez-Adan Transgenesis in large domestic species: future development for milk modification. Reprod. Nutr. Dev. 39: Soukharev S., D. Hammond, N. M. Ananyeva, J. A. Anderson, C. A. Hauser, S. Pipe and E. L. Saenko Expression of factor VIII in recombinant and transgenic systems. Blood Cells Mol. Dis. 28: Takahashi R. and M. Ueda The milk protein promoter is a useful tool for developing a rat with tolerance to a human protein. Transgenic Res 10: Wu, H. T. and M. C. Huang Effects of electroporation on the quality of rooster semen. AJAS 8:

10 DNA DNA 4

11 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

12 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

13 DNA bovine papilloma virus, BPV DNA 8,000 BPV DNA DNA plasmid DNA DNA DNA insulin BPV DNA DNA mrna BPV DNA pbr 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

14 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 µm microinjection micropipette puller micromanipulator DNA 3. DNA DNA 4. DNA DNA lipid bilayer 8

15 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 Hommond Whitten 9

16 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

17 DNA 30% 10% DNA 3. DNA transfect DNA DNA DNA 5. 11

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

19 **** 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

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21 1993 Bioindustry 4: : : Bioindustry 9:1-9 Boyton, R. J. and D. M. Altmann Transgenic models of autoimmune disease. Clin. Exp. Immunol. 127:4-11. Finn, O. J. and G. Forni Prophylactic cancer vaccines. Curr. Opin. Immunol. 14: Greten, F. R., M. Wagner, C. K. Weber, U. Zechner, G. Adler and R. M. Schmid TGF alpha transgenic mice: a model of pancreatic cancer development. Pancreatology 1: Hard, G 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 Molecular mechanism of viral hepatocarcinogenesis. Oncology 62: Kozak, L. P. and M. Rossmeisl Adiposity and the development of diabetes in mouse genetic models. Ann. N. Y. Acad. Sci. 967: Lamartiniere, C. A., M. S. Cotroneo, W. A. Fritz, J. Wang, R. Mentor-Marcel and A. Elgavish Genistein chemoprevention: timing and mechanisms of action in murine mammary and prostate. J. Nutr. 132:552S-558S. McClain, D. A Hexosamines as mediators of nutrient sensing and regulation in diabetes. J. Diabetes Complications 16: Olgin, J. E. and S. Verheule Transgenic and knockout mouse models of atrial arrhythmias. Cardiovasc. Res. 54: Szalai, A. J The antimicrobial activity of C-reactive protein. Microbes. Infect. 4: Zaugg, M., M. C. Schaub, T. Pasch and D. R. Spahn Modulation of beta-adrenergic receptor subtype activities in perioperative medicine: mechanisms and sites of action. Br. J. 15

22 Anaesth. 88:

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

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

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

26 terminization congression sister chromatids interphase polar body interkinesis DNA 20

27 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

28 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

29 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 A A16 A32 A1 A1 A2 A3 A4 B preleptotene primary 23

30 spermatocyte sertoli cells epididymis motile sperm golgi phase cap phase acrosomal phase maturation phase acrosomal granules headtail flagellum acrosome ATP microtubules central filaments 9 24

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

32 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

33 pronucleus female pronuclei syngamy zygote cleavage blastomeres cell mass hatch RNA inactive totipotency polarization inncer cell mass, ICM trophectoderm 27

34 the emergence of pattern change in form 28

35 ( ) ( ) 2n 4n 2C DNA n+n a. b. a. b. 29

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

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

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

39 Campell, N. A. and J. B. Reece Biology. 6 th ed., pp , Benjamin Cummings San Francisco, USA. Garner, D. L. and E. F. E. Hafez Spermatozoa and seminal plasma. In: Reproduction in farm animals. 7 th ed., pp Lippincott Williams & Wilkins, Philadelphia, USA. Hafez, E. S. E. and B. Hafez Folliculogenesis, egg maturation, and ovulation. In: Reproduction in Farm Animals. 7 th ed., pp 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 Spermatogonial stem cell transplantation. Mol. Cell Endocriol. 169: Karp, G Cell and molecular biology: Concepts and Experiment. 2 nd ed., pp John Wiley & Sons, NY, USA. McLean, D. J., D. S. Johnston, L. D. Russell, M. D. Griswold Germ cell transplantation and the study of testicular function. Trends Endocrinol. Metab. 12: Ogawa, T Spermatogonial transplantation: the principle and possible applications. J. Mol. Med. 79: Senger, P. L Pathways to pregnancy and parturition. 1 st ed., pp , pp , pp Current Conceptions, WA, USA. Ventela, S, H. Ohta, M. Parvinen, Y. Nishimune Development of the stages of the cycle in mouse seminiferous epithelium after transplantation of green fluorescent protein-labeled spermatogonial stem cells. Biol. Reprod. 66: Wolpert, L., R. Beddington, J. Brockes, T. Jessesll, P. Lawrence and E. Meyerowitz Principles of development. pp Current Biology Ltd, London. 33

40 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

41 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

42 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

43 1 hormones FSH LH releasing hormone, LH-RH LH PGE2 PGF2α 2 cyclic adenosine 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

44 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

45 IVM hr M M NCSU 23, M M-199, F10, F M2, HTF 24 MII cytoplasmic maturation FSH LH 80 90% 40 80% COCs 1. M mm µl 3 4 ml 2 COCs IVM 5% CO

46 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 % FCS Added to 20 ml ROSI, ICSI, NT GV GV IVM KSOM HTF KSOM Component Concentration, mm g/l NaCl KCl KH 2 PO MgSO Na lactate Glucose Na pyruvate NaHCO CaCl 2 2H 2 O Gutamine EDTA BSA 1.0 mg/ml Penicillin-G 100 U/mL 60U/mL Streptomycin sulfate 50 U/mL Modified from Biggers et al

47 HTF* Component mm NaCl KCl 4.69 MgSO KH 2 PO Na lactate 1.32 g/ml 21.4 Glucose 2.78 CaCl 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 cortical granule reaction zona reaction Ca 2+ activation Ca 2+ smooth endoplasmic reticulum Ca Miyazaki 1990 Ca 2+ Ca 2+ Miyazaki receptor-mediated 41

48 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

49 MII capacitation 20 cumulus cell mass ph 70% 12 ml 3.5 cm % 12 ml BO-washing medium 7 ml BO-washing medium 3000 rpm ml BO-washing medium 43

50 BO medium ml BO medium 10 6 /ml X BO medium, 10X Chemicals Weight, g Concentration, mm NaCl KCl NaH 2 PO 4 H 2 O Glucose Penicillin Streptomycin Phenol red 0.2% 4 ml 4% MgCl 2 6H 2 O CaCl 2 2H 2 O Milli Q water Added to 100 ml 100 ml 5. BO-fertilization medium 2 50 µl/droplet COCs BO-fertilization medium COCs 50 µl 50 µl 5% CO

51 BSA KSOM potassium simplex optimized medium M-199 b synthetic oviductal fluid mm pyruvatelactate glutamine taurine 20 transgenic animal 45

52 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

53 Biggers, J. D., L. K. McGinnis and M. Raffin Amino acids and preimplantation development of the mouse in protein-free potassium simplex optimized medium. Biol. Reprod. 63: Brackett, B. G. and G. Oliphant Capacitation of rabbit spermatozoa in vitro. Biol. Reprod. 12: Choi, T., F. Aoki, M. Mori, M. Yamashita, Y. Nagahama and K. Kohmoto Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos. Development 113: Eppig, J. J 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: Ju, J. C., J. E. Parks and X. Yang Thermotolerance of IVM-derived bovine oocytes and embryos after short-term heat shock. Mol. Reprod. Dev. 53: Mattioli, M., G. Galeati, M. L. Bacci and B. Barboni Changes in maturation-promoting activity in the cytoplasm of pig oocytes throughout maturation. Mol. Reprod. Dev. 30: Miyazaki, S 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: Perry, A. C. F., T. Wakayama, I. M. Cooke and R. Yanagimachi Mammalian oocyte activation by the synergistic action of discrete sperm head components: induction of calcium transients and involvement of proteolysis. Dev. Biol. 217: Stice, S. L. and J. M. Robl Activation of mammalian oocytes by a factor obtained from rabbit sperm. Mol. Reprod. Dev. 25: Watanabe, N., G. F. Vande Woude, Y. Ikawa and N. Sagata Specific proteolysis of the c-mos proto-oncogene product by calpain on fertilization of Xenopus egg. Nature 342:

54 1980 DNA DNA gene therapy 48

55 10 kb DNA position effect DNA DNA DNA BglII KpnI 20 Seo recombinant DNA SV40 DNA SV40 DNA 49

56 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

57 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

58 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

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

60 1966 Pain polymerase chain reaction, PCR Southern blot hybridization 54

61 gain of function loss of function 55

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

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

64 retrovirus % 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

65 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

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

67 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

68 inner cell mass, ICM mosaic germline transmission rate 10% pl 10 8 cfu/ml PSNam 10% FCS DMEM DMEM-10 70% µm 25,500 g /100 DMEM µm -80 PSNam DMEM % DMEM µm 25,500 g /100 DMEM µm Chan DNA 293T T-75 CMV/VSV-G µm 50,000 g µl Hank s

69 ph VSV-G ph % ph ph 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% % 8 10 ml

70 pl cfu/ml 25,500 g DMEM-10 M16 DMEM 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

71 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

72 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

73 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

74 ( ) 68

75 : Andreadis, S. and B. O. Palsson Coupled effects of polybrene and calf serum on the efficiency of retrovial transduction and stability of retroviral vectors. Human Gene Therapy 8: Chan, A. W. S., E. J. Homan, L. U. Ballou, J. C. Burns and R. D. Bremel Transgenic cattle produced by reverse-transcribed gene transfer in oocytes. Proc. Natl. Acad. Sci. USA 95: Chan, A. W. S., K. Y. Chong, C. Martinovich, C. Simerly and G. Schatten Transgenic monkeys produced by retroviral gene transfer into mature oocytes. Science 291: Jaenisch, R Germ line integration and Mendelian transmission of the exogenous moloney leukemia virus. Proc. Natl. Acad. Sci. USA 73: Jaenisch, R., H. Fan and B. Croker 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: Lois, C., E. J. Hong, S. Pease, E. J. Brown and D. Baltimore Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295: Miller, O. G., M. A. Adam and A. D. Miller Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol. Cell Biol. 10: Mizuarai, S., K. Ono, K. Yamaguchi, K. Nishijima, M. Kamihira and S. Iijima Production of transgenic quails with high frequency of germ-line transmission using VSV-G pseudotyped retroviral vector. Biochem. Biophys. Res. Commun. 286: Peters, R. M., B. H. Johnson and R. M. Shuman Gene transfer in swine embryos using an avian retrovirus. J. Cell. Biochem., Suppl. 13B:182, Abstract. 69

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

77 RNAi RCAS-TVA RNA RNA interference, RNAi RNA mrna RNA Dicer 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

78 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

79 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 sirna, small interfering RNA sirna RNA RNA RNAi RNA RNAi sirna DNA 73

80 RNA insert DNA 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 S0209 RNAi RNAi RNA RNA mrna RNAi RNA RNAi RNAi RNA RNAi RNA RNAi RNAi transposon RNAi RNAi (C. elegans) RNA 74

81 Initiation step RNA trigger Dicer 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 mer sirna sirna mrna mrna RNA RNA dependent RNA polymerase, RdRP RNA 75

82 RNA Dicer 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

83 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

84 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

85 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

86 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

87 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

88 TVA RCAS-TVA 1. RACS-TVA TVA 2. RACS-TVA gene of interest 3. TVA 4. RCAS-cDNA ( b/tva2.html) 5. RCAS 6. TVA 82

89 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

90 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

91 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

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

93 RCAS ( gene X ) ClaI "Reprinted from 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 hivdrp/rcas/ by permission of the HIV Drug Resistance Program, National Cancer Institute" 87

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

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

96 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 DNA DNase DNA DNA DNA 32 P DNA 39% 355/913 DNA equatorial segment 1% DNA 90

97 DNA DNA DNA DNA % DNA DNA 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

98 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 % DNA PCR DNA 92

99 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

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

101 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

102 96

103 DNA DNA Southern 97

104 : DNA 24: Atkinson, P. W., E. R. Hines, S. Beaton, K. I. Matthaei, K. C. Reed and M. P. Bradley 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 No simple solution for making transgenic mice. Cell 59: 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 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 Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. USA 77: 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 Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315: Lavitrano, M., A. Camaioni, V. M. Fazio, S. Dolci, M. G. Farace and C. Spadafora Sperm cells as vectors for introducing foreign DNA into eggs: Genetic transformation of mice. Cell 57: Lavitrano, M., D. French, E. Forte, M. Francolini, S. Sperandio, R. Testi and C. Spadafora The interaction of sperm cells with exogenous DNA: a role of CD4 and major histocompatibility complex class II molecules. Exp. Cell Res. 233: Zani, M., M. Lavitrano, D. French, V. Lulli, B. Maione, S. Sperandio and C. Spadafora The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake. Exp. Cell Res. 217:

105 human decay accelerating factor, hdaf 99

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

107 1980 HSV herpes simplex virus SV40 simian virus 40 DNA 5 15% DNA rat mouse mmt-hgh DNA Lavitrano % 50 60% DNA DNA 101

108 reprogram 1997 Wilmut Dolly PPL hfix Polly 2002 hfix 0.2 2% kb embryonic stem cells, ES cells inner cell mass, ICM 102

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

110 DNA DNA DNA Banding DNA TE 10 mm Tris/0.1 mm EDTA, ph= ng/µl GMO DNA DNA 0.22 µm µl PGF2α PGF2α 2. regumate 0.4% Altrenogest 20 mg PGF2α PMSG HCG PMSG 24 HCG PMSG HCG

111 HCG HCG HCG 6: HCG 8: PMSG/HCG Intervet 1500/1250 IU2000/1750 IU 500/500 IU 5 6 PMSG/HCG PMSG/HCG 7 8 FDA 105

112 HCG HCG cm 25 ml D-PBS 0.1% BSA / 2. 5 cm 1 cm ml D-PBS 0.1% BSA / ml 106

113 15,000 23,500 g µm mm DNA 0.6 µl DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA HCG HCG

114 DNA DNA DNA HCG germinal vesicle, GV PMSG HCG GV PMSG/HCG 1 2 PGF2α Kliev s

115 DNA DNA PCR PCR PCR nitrocellulose membrane DNA NC 32 P-dCTP gene 109

116 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

117 DNA HPLC mrna hemizygote 50% DNA F1 F2 PMSG/HCG

118 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

119 113

120 : : : (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

121 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 Production of transgenic mice, rabbits and pigs by microinjection foreign DNA into pronuclei. Zuchthygiene 20: Brinster, R. L., H. Y. Chen, M. E. Trumbauer, M. K. Yagle and R. D. Palmiter Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. USA 82: 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 Transgenic pigs producing in vitro matured oocytes infected with a retroviral vector. Anim. Biotech. 12: Gorden, J. W., G. A. Scangos, D. J. Plotkin, J. A. Barrosa and F. H. Ruddle Genetic transformation of mouse embryos by microinjection od purified DNA. Proc. Natl. Acad. Sci. USA 77: 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 Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315: Kupriyanov, S., K. Zeh and H. Baribault Double pronuclei injection of DNA into zygotes increases yields of transgenic mouse lines. Transgenic Res. 7: Lavitrano, M., A. Camaioni, V. M. Fazio, S. Dolci, M. G. Frace and C. Spadafora Sperm cells as vector for introducing foreign DNA into eggs: genetic transformation of mice. Cell 57: 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 Sperm-mediated gene transfer: production of pigs transgenic for a human regulator of complement activation. Transplant Proc. 29: Lin, T. P Microinjection of mouse embryo. Science 151: Martin, M. J. and C. A. Pinkert Production of transgenic swine. In: Transgenic Animal Technology: A Laboratory Handbook, pp 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 Dramatic 115

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

123 Genzyme Transgenics 60 US$ US$ cents 30,000 60,000 DNA DNA 117

124 250 3 g 400 mg IgY TranXenoGen AviGenics DNA primordial germ cells, PGC Eyal-Giladi Kochav I-XIV Hamburger Hamilton 1 46 Eyal-Giladi Kochav X stage X area pellucida area opaca epiblast 118

125 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

126 II III % III 40% II III 20% Naito I II 30% DNA DNA DNA DNA

127 20 60, µm DNA PGC DNA 0.1 mg/ml 3 nl nl lacz gene β-actin-lacz % 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

128 PGC PGC germline chimera PGC DNA PGC PGC Hamburger Hamilton PGC PGC PGC PGC PGC PGC DNA transfection DNA PGC DNA PGC PGC PGC PGC PGC DNA PGC 122

129 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) % 32(22) 20 19/20 (95) 2-96% Incubation Original egg shell incubation Ex vivo incubation Ref. Chang, et. al., 1997 Naito, et. al., Circular PGCs 83(26) 74 31/74(42) % *donor-de.%: Percentage of chicks with donor-derived plumage Ex vivo incubation Naito, et. al., 1999 blastomeres stage X 2 DPBS hairloop CMF-PBS % trypsin 5 10 DMEM 10% FBS PGC 5 15% ex vivo 30% 123

130 I I i i I I i DNA DNA % 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 (30) 50(4.9) 13(26) 3/13(23) % Recipient γ irradiation Thoraval et. al., (35) 52(7.3) 23(44) 0 No γ irradiation (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

131 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

132 TMGT testis-mediated gene transfer DNA DNA lipofection 126

133 I 24 II III 127

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

135 Chang, I., D. K. Jeong, Y. H. Hong, T. S. Park, Y. K. Moon, T. Ohno and J. Y. Han Production of germline chimeric chicken by transfer of cultured chick primordial germ cells. Cell Biol. Int. 21: Eyal-Giladi, H. and S. Kochav 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: Hamburger, V. and H. L. Hamilton A series of normal stages in the development of the chick embryo. J. Morph. 88: Kagami, H., T. Tagami, Y. Matsubara, T. Harrumi, H. Hanada, K. Maruyama, M. Sakurai, T. Kuwana and M. Naito 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: Love, J., C. Gribbin, C. Mather and H. Sang Transgenic birds by DNA microinjection. Biotechnology 12: Naito, M., A. Tajima, Y. Yasuda and T. Kuwana Production of germline chimeric chickens, with high transmission rate of donor-derived gametes, produced by transfer of primordial germ cells. Mol. Reprod. Dev. 39: Naito, M., K. Agata, K. Otsuka, K. Kino, M. Ohta, K. Hirose, M. M. Perry and G. Eguchi Embryonic expression of β-actin-lacz hybrid gene injected into the fertilized ovum of the domestic fowl. Int. J. Dev. Biol. 35: Naito, M., Y. Matsubara, T. Harrumi, T. Tagami, H. Kagami, M. Sakurai and T. Kuwana 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: Nakanishio, A. and A. Iritani Gene transfer in the chicken by sperm-mediated methods. Mol. Reprod. Dev. 36: Perry, M. M A complete culture system for the chick embryo. Nature 331: Sang, H Transgenic chickens- methods and potential applications. Trends in Biotechnology. 12: Speksnnijder, G. J., R. J. Etches and A. M. V. Gibbins Germline chimeric chickens from FACS-sorted donor cells. Mol. Reprod. Dev. 52: Thoraval, P., F. Lasserre, F. Coudert and G. Dambrine

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

137 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

138 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 kda 1979 chromophore 1996 GFP 3-dimentional structure Prasher 1992 wt GFP 1994 GFP 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

139 24 wt GFP ph EGFP ph 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 nm Stokes shift 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

140 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 magenta 134 cyan

141 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/ / emission 477/ / ( ) (absorbance )extinction ~ coefficient (,10 3 M -1 cm -1 ) quantum yield (QY,, %) Patterson 2001Clontech absorbance, A A/bc b cm c /L intensity x QY a b wt GFP regulatory sequence GFP GFP GFP 135

142 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 Okabe pcx-egfp microinjecting pronucleus 136

143 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 nm line spectrum nm 10 nm background emission HBO 50 W/AC arc size mm 200 V 2,000 lumens, lm 30,000 candela/cm 2, cd/cm W/AC arc size mm 45 V 2,200 lm 170,000 cd/cm

144 2. high-pressure xenon short arc lamp nm continuous spectrum XBO 75 W/2 arc size mm DC V 1,000 lm 40,000 cd/cm absorption filter nm transmittance glass, G colored filter 520 nm Zeiss G nm 2. band-pass BP interference filter 10 nm 138

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

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

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

148 ( ) (nm) 142

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

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

151 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

152 12 40% ( ) Acanthopagrus schlegeli Penaeus monodon Macrobrachium rosenbergii GH GH GH copies GH GH cdna GH cdna g g g 13 50% DNA

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

154 3 5 cm, 148

155 WTO American Fisheries Society DNA 149

156 1995 Frankenfish Salmo salar Aqua Bounty Farms 1999 coho salmon Oncorhynchus kisutch coho salmon

157 in vivo 151

158 6 ( ) 2.5 ( ) ( ) 6 ( ) DNA 152

159 DNA ( ) DNA DNA 153

160 Hsiao, C. D., F. J. Hsieh and H. J. Tsai Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish. Dev. Dynam. 220: Li, S. S. and H. J. Tsai Transfer of foreign gene to giant freshwater prawn Macrobrachium rosenbergii by spermatophore- microinjection. Mol. Reprod. Dev. 56: Lu, J. K., T. T. Chen, C. L. Chrisman, O. M. Andrisani and J. E. Dixon Integration, expression, and germ-line transmission of foreign growth hormone genes in medaka Oryzias latipes. Mol. Marine Biol. Biotechnol. 1: Ma, G. C., T. M. Wang, C. Y. Su, Y. L. Wang, S. Chen and H. J. Tsai Retina-specific cis-elements and their binding nuclear proteins of carp rhodopsin gene. FEBS Lett. 508: Tsai, H. J., C. H. Lai and H. S. Yang Sperm as a carrier to introduce an exogenous DNA fragment into the oocyte of Japanese abalone Haliotis disvorsicolor suportexta. Transgenic Res. 6: Tsai, H. J. and F. S. Tseng Electroporation of a foreign gene into black porgy Acanthopagrus schlegeli embryos. Fish. Sci. 60: 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: 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 Introducing foreign DNA into tiger shrimp Penaeus monodon by electroporation. Theriogenology 54: Tseng, F. S., I. C. Liao and H. J. Tsai Transient expression of mouse tyrosinase gene in catfish albino mutants Clarias fuscus by subcutaneous microinjection. Fish. Sci. 61:

161 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

162 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

163 C57BL/6 BALB/C rat targeting vector isogenic genomic DNA library λ λ phage, kb cosmid 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

164 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

165 reporter gene β-galactosidase lacz electroporation ori kb 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

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

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

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

169 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

170 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

171 +/- 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

172 The Jackson Laboratory Cre 166

173 (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

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

175 (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

176 (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

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

178 gene targeting gene knockout Dolly totipotency 1997 Polly

179 fibroblast α-1, Briggs King 2n 1986 Willadsen 173

180 1989 Prather Wilmut G0 -Dolly 1997 Schnieke -Polly 2000 Roslin Institute PPL α-1, Lai PPL Dai Infigen Genmark

181 chimera mm % 175

182 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 % 10% 30% Park 2001 Lai 2002 α-1,3- PPL 176

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

184 4 0.5% " fetal cells Infigen Genmark Prairie State Semen Inc. "The man" "401K" 178

185 Infigen 1992 Chen % % % 6/ % µg/ml 5 µg/ml

186 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

187 α-gt % C 2000 Dolly PPL α-1, Lai PPL Dai 181

188 1997 PPL telomere " " 182

189 embryo cloning 183

190 " " " " 184

191 185

192 ( ) 186

193

194 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 Production of cloned pigs from in vitro systems. Nature Biotechnol. 18: 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 Targeted disruption of the α1,3-galactosyltransferase gene in cloned pigs. Nature Biotechnol. 20: 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 Production of α-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295: Onishi, A., M. Iwamoto, T. Akita, S. Mikawa, K. Takeda, T. Awata, H. Hanada and A. C. F. Perry Pig cloning by microinjection of fetal fibroblast nuclei. Science 289: 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 Production of nuclear transfer-derived swine that expressed the enhanced green fluorescent protein. Anim. Biotechnol. 12: 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 Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407: Prather, R. S., M. M. Sims and N. L. First Nuclear transplantation in early pig embryos. Biol. Reprod. 41: 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 Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278: Wilmut, I., A. E. Schnieke, J. McWhir, A. J. Kind and K. H. S. Campbell Viable offspring derived from fetal and adult mammalian cells. Nature 385:

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

196 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

197 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

198 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

199 Southern blot hybridization 2 10 A HPV18/LCR SV40/TAg DNA slot-blot 18LT-12 18LT-28 18LT LT-11 18LT 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

200 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 '-SmaI 5'-XbaI 16.7% vs 15.4% DNA bp 3'- 80.1% vs 61.6% 200 bp 5'- 3'- 23.1% vs 3.3% 5 overlapping 61.3% 19/ "TCCC" DNA 194

201 Transgenic Transgene Nucleotide deletion a Insertion mouse junction Left tg Overlapping Right tg Total sequnce (bp) sequnce (bp) (bp) ( )3-5 junction ( type): 18LT LT ATCT 18LT TGGATT LT TCCC LT LT ACCTAA 18LT-31 5 I.d. GT I.d LT TCCC LT C LT TGTGG LT TCCC LT TT 18LT GCAA LT LT I.d. -- I.d LT LT CGACT 18LT TCCC LT GGAT LT TTGC LT AC LT CGACT ( ) 3-3 junctions ( type): 18LT ACAAA LT CA LT LT TTA LT CA LT ( ) 5-5 junctions ( type): 18LT ATT LT GAT LT AAA ( ) Transgene-cellular junctions ( type): 18LT *******Cellular sequence******* 18LT *******Cellular sequence******* 18LT *******Cellular sequence******* 18LT *******Cellular sequence******* 18LT *******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

202 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 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

203 hypervariable minisatelite 10 DNA motifs No. of seq. with motif Motif / no. analyzed Frequency (%) (A) Single motif: Topo motifs 70/ Topo * motifs 45/ Homo-R or homo-y 39/ Alternating R-Y 26/ (A-T)-rich 21/ (B) Motifs coincidental with Topo * sites : Homo-R or-y/topo * 33/ Alternating R-Y/Topo * 14/ (A-T)-rich/Topo * 14/ Homo-R or-y/alt R-Y/Topo * 9/ Homo-R or-y/(a-t)-rich/topo * 8/ LCR/TAg :3 DNA DNA 197

204 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

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

206 Chen, C. M., C. H. Wang, S. C. Wu, C. C. Lin, S. H. Lin and W. T. K. Cheng 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: Chen, C. M., W. T. K. Cheng and K. B. Choo 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: Derbyshire, M. K., L. H. Epstein, C. S. Young, P. L. Munz and R. Fishel Nonhomologous recombination in human cells. Mol. Cell. Biol. 14: Eisenstark, A Genetic recombination in bacteria. Ann. Rev. Genet. 11: Holliday, R A mechanism for gene conversion in fungi. Genet. Res. 5: Konopka, A. K Compilation of DNA strand exchange sites for non-homologous recombination in somatic cells. Nucleic Acids Res. 16: Moser, H. E. and P. B. Dervan Sequence-specific cleavage of double helical DNA by triple helix formation. Science 238: Nicolas, A. L. and C. S. Young 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: Pfeiffer, P., S. Thode, J. Hancke and W. Vielmetter Mechanisms of overlap formation in nonhomlogous DNA end joining. Mol. Cell. Biol. 14: Roth, D. B., G. N. Proctor, L. K. Stewart and J. H. Wilson Oligonucleotide capture during end joining in mammalian cells. Nucleic Acids Res. 19:

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

208 DNA 1 2 ng/µl DNA 2 ng/µl 10 ng/µl ng/µl DNA ng/µl 0.5 ng/µl DNA holding pipette injection needle µm 20 µm 4 µm DNA DNA DNA DNA DNA DNA 202

209 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

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

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

212 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 NFI NF1 2. STAT5 STAT5 5 -ACTTCTTGGAATT TTCTTGGAA-3 STAT5 DNA mammary gland factor, MGF milk protein binding factor, MPBF STAT5 STAT5a, b 93% 6 MGF 21 STAT5a, b STAT5 206

213 STAT5 STAT5 DNA STAT5 3. C/EBP C/EBP 7 β 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 ; 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

214 β- 5' - C/EBPb YY1 β- 6. OCT1 αs2-5' OCT ' -50 OCT

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

216 : : 37: : 30:45-48 Brink M. F., M. D. Bishop and F. R. Pieper Developing efficient strategies for the generation of transgenic cattle which produce biopharmaceuticals in milk. Theriogenology 53: Houdebine L. M The methods to generate transgenic animals and to control transgene expression. J. Biotechnol. 98: Houdebine L. M., S. Rival, T. Pantano, G. Jolivet, D. Thepot and J. Attal Transgenesis for the study and the control of lactation. Reprod. Nutr. Dev. 42: Huang, M. C. and J. S. Chao Regulatory sequences of the 5' flanking region of goat β-casein gene. AJAS 14: Pintado B. and A. Gutierrez-Adan Transgenesis in large domestic species: future development for milk modification. Reprod. Nutr. Dev. 39: Salaheddine M., E. Thole and M. Brink Transgenic cattle constitute a breakthrough in production of innovative medicine. From milk to medicine. Tijdschr Diergeneeskd. 127: 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 Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nat. Biotechnol. 20:

217 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

218 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

219 GHRH IGF-1 IGF-1 DNA IGF-1 Pursel IGF-1 α-actin promoter IGF-1 IGF-1 IGF 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

220 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

221 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

222 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

223 1997 Wilmut nuclear transfer 217

224 Bawden, C. S., A. V. Sivaprasad, S. K. Verma, S. K. Walker and G. E. Rogers 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: Brophy, B., G. Smolenski, T. Wheeler, D. Wells, P. L Huillier and G. Laible Cloned transgenic cattle produce milk with higher levels of β-casein and κ-casein. Nat. Biotechnol. 21: Devlin, R. H., T. Y. Yesaki, E. M. Donaldson, S. J. Du and C. L. Hew Production of germline transgenic Pacific salmonids with dramatically increased growth performance. Can. J. Fish Aquat. Sci. 52: Gutierrez-Adan, A., E. A. Maga, H. M. Meade, C. F. Shoemaker, J. F. Medrano, G. B. Anderson and J. D. Murray Alteration of physical characteristics of milk from bovine kappa-casein transgenic mice. J. Dairy Sci. 79: 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 Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hdaf) transgenic pigs for xenotransplantation. Proc. Natl. Acad. Sci. USA 99: Müller, M., B. Brenig, E. L. Winnacker and G. Brem Transgenic pigs carring cdna copies encoding the murine Mx1 protein which confers resistance to influenza virus infection. Gene 121: Palmiter, R. D., R. L. Brinster, R. E. Hammer, M. E. Trumbauer, M. G. Rosenfeld, N. C. Birnberg and R. M. Evans Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth fusion gene. Nature 300: Pinkert, C. A The history and theory of transgenic animals. Lab. Anim. 26: Tu, C. F., K. Tsuji, K. H. Lee, R. Chu, T. J. Sun, Y. C. Lee, C. N. Weng, C. J. Lee Generation of HLA-DP transgenic pigs for study of xenotransplantation. Int. Surg. 84: Wilmut, I., A. E. Schneike, J. McWhir, A. J. Kind and K. H. Campbell Viable offspring derived from fetal and adult mammalian cells. Nature 385:

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

226 post genome world % embryonic stem ES cell 220

227 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

228 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

229 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

230 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

231 1 ENU ENU mutagenesis screens ENU N-ethyl-N-nitrosourea 苷 ethyl DNA DNA F1 F1 founder F2 F2 F1 founder backcross F3 2 gene trap ES 225

232 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

233 dominant negative alleles ENU 1. 1 α1-antitrypsin 227

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

235 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

236 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

237 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 kb insulator Insulator DNA insulator 5 HS4 chromatin opener 5 HS4 231

238 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

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

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

241 1 ml ScFv Fab Fab IgA IgA α-lactalbumin prion [ 1 4 ] cm 235

242 236

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

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

245 gametes embryo /

246 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

247 seeding PBS PBS /

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

249 M 1.0 M 0.8 M cryopreservation freezing 2 3 recrystallization 243

250 4 1.5 M DMSO 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 / phase hypertonic organelles 244

251 / / M EG Ethylene Glycol0.4% BSA 0.1 M sucrose DPBS 245

252 EG M EG Agca, Y., R. L. Monson, D. L. Northey, O. A. Mazni, D. M. Schaefer and I. J. Rutledge Transfer of fresh and cryopreserved IVP bovine embryos: Normal calving, birth weight and gestation lengths. Theriogenology 50: Eppig, J. J. and M. J. O Brien Development in vitro of mouse oocytes from primordial follicles. Biol. Reprod. 54: Lehn-Jensen, H. and S. M. Willadsen Deep-freezing of cow half and quarter embryos. Theriogenology 19: Leibo, S. P. and N. M. Loskutoff Cryobiology of in vitro-derived bovine embryos. Theriogenology 39: 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 The University of Sydney: Proceedings No. 70. Pedersen, R. A., A. McLaren and N. L. First 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 Cryopreservation of in vitro produced bovine embryos: 246

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

254 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

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

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

257 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 M K d 10-4 M K d 10-8 M ligand matrix spacer arm 251

258 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

259 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

260 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

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

262 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

263 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

264 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

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

266 ph ph 260

267 Citric acid Lactic acid Butanedioic acid Acetic acid Malonic acid MES Phosphate HEPES BICINE ph N-methylpiperazine Piperazine bis-tris bis-trispropane Triethanolamine Tris N-methyldiethanolamine 1,3-diaminopropane ethanolamine Piperazine 1,3-diaminopropane Piperazine ph 261

268 SDS- PAGE N- 262

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

270

271 exogenous gene endogenous gene biohazard 1. transgene 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

272 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

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

274 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

275 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

276 barrier facility embryo transfer 3. C57BL/6 FVB 4. transmissible spongiform encephalopathy TSE cm 270

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

278 3 10. refinement DNA. 3 4 phenotype 272

279 5 6 standard operation procedure, SOP 7 end point Transgenic Sheet Information Animal Care Committee Institutional Biohazard Committee end point CO 2 273

280 ILAR 1992 interspecies committee 1. transgene TgX(YYYYY)#####Zzz 1 TgX 274

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

282 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 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

283 founder 2 founderc57bl/6 C57BL/6 99% C57BL/6 C57BL/6 congenic strain C57BL/6 C57BL/6 congenic strain 129 C57BL/ C57BL/ congenic strain congenic strain microsatellite simple sequence length polymorphisms SSLP nucleotide repeats PCR primer pairs C57BL/ C57BL/6 congenic strain 21 WTO 277

284 278

285 279 congenic strain

286 pp Animal Welfare Act USDA: pubs/awact.html Genetic engineering: animal welfare and ethics A discussion paper from the Boyd Group. Guidelines for Research Involving Recombinant DNA Molecules NIH Guidelines. Hessler, J. R., J. R. Broderson and C. S. King Rodent quarantine: facility design and equipment for small animal containment facilities. Lab. Animal 28: Joyner, A. L Gene Targeting: a Practical Approach. Oxford press. Marie, B Transgenic Animals, Animal Welfare and Ethics. Canadian Council on Animal Care (CCAC). Marie, H. L Transgenic Animals: Generation and Use. pp Harwood Academic Publishers. Muriel, T. D Guidelines for Naming Transgenic Mice. Lab. Animal. The Report and Recommendations of ECVAM Workshop 28 1, The Use of Transgenic Animals in the Europe Union. ATLA 26:

287 pp DNA DNA P1 P2 1. LS-1 LS-1 DNA DNA A. DNA DNA DNA B. DNA DNA a b 281

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

289

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

291 1. Heterozygotes Homozygote

292

293 pregnant mare s serum gonadotropin, PMSG human chorionic gonadotropin, HCG HCG LH PMSG HCG LH PMSG HCG vaginal plug h 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

294 5 10 IU PMSG a, b HCG 5 10 IU/ HCG c 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,

295 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

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

297 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 % CO 2 95% 100% 5% O 2 5% CO 2 90% N

298 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 ml 200 ml ml Milli-Q H 2 O 2. BSA BSA 291

299 5. BSA 6. 1 ml pipette 5% O 2 5% CO 2 90% N ph mosm 1. plug 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 blastocyst hatching

300 2-cell stage embryo 4-cell stage embryo 8-cell stage embryo morula blastocyst hatching 293

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

302 Component (mg/100 ml) MWM M16 CZB KSOM NaCl KCl CaCl 2 2H 2 O KH 2 PO MgSO 4 7H 2 O NaHCO Glucose Sodium lactate or mg of 60% 530 µl (60% syrup) 174 µl (60% syrup) syrup Calcium lactate 46.0 Sodium pyruvate Glutamine Streptomycin sulfate Penicillin G potassium salt Phenol Red 100 µl L Mercaptoethanol, 20 mm EDTA-2Na (1% solution) 50 µl 50 µl (100 mm) Ess. Amino acid (50X) 1000 µl Non-ess. Amino acid (100X) 500 µl BSA

303 Component (mg/100 ml) PBI M2 NaCl KCl CaCl CaCl 2 2H 2 O 25.2 KH 2 PO MgCl 2 6H 2 O 10.0 MgSO 4 7H 2 O 29.3 Na 2 HPO NaHCO HEPES Glucose Sodium pyruvate Sodium lactate or mg of 60% syrup Penicillin G potassium salt Streptomycin sulfate 5.0 Phenol Red 1.0 BSA

304 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

305 DNA DNA RNA DNA DNA DNA DNA DNA DNA DNA DNA ml 2. 5 ml 500 ml ,000 rpm ml Solution I ml Solution II ml Solution III , 12,000 rpm

306 % EtOH M NaOAc DNA ,000 rpm % EtOH DNA DNA 13. DNA 11 ml TE buffer g CsCl CsCl ml EtBr ,000 rpm µl Triton ,000 rpm DNA 22. buffer-saturated butanol DNA EtBr 23. DNA dialysis cassette 16 DNA 1. DNA 2. DNA % 1.5% LMP 0.5 g/ml EtBr 4. DNA LMP 5. LMP gel LMP M NaCl 5 8. phenol 299

307 9. 10,000 rpm phenol 11. chloroform DNA % EtOH M NaOAc DNA ,000 rpm % EtOH DNA DNA 16. DNA TE buffer DNA 1. DNA 3 ml 3 5 g DNA 2. 3g CsCl CsCl 1 g CsCl/mL L EtBr ,000 rpm L TritonX ,000 rpm DNA 9. buffer-saturated butanol DNA EtBr 10. dialysis cassette 16 buffer

308 (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

309 Chen, C. M., K. B. Choo and W. T. K. Cheng 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: Sambrook, J., E. F. Fritsch and T. Maniatis Molecular Cloning: A Laboratory Manual, 2 nd ed., pp Cold Spring Harbor Laboratory Press. New York, USA. 302

310 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 cm puller, Narishige PN-3, Japan microforge, Narishige MF-9, Japan µm µm Sutter Instrument Co., Model P

311 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 µm burn polish mouth pipette DNA M

312 M2 M2 ( Hogan et al., ) 305

313 : pp Hogan, B., R. Beddington, F. Costantiti and E. Lacy Manipulating the Mouse Emryo, 2 nd ed., pp , Cold Spring Harbor Laboratory Press, NY, USA. 306

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

315 2.5% avertin 3% pentobarbital ml/g a 1 ml 27G 3 4 2,2,2-tribromoethanol 1. autoclaved 2. a 3. 75% b cm b 5. fat pad, c 6. d, e f

316 a b c 309

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

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

319 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

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

321 314

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

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

324 Gordon, J. W Production of transgenic mice. Methods Enzymol. 225: Gordon, J. W. and F. H. Ruddle Gene transfer into mouse embryos: Production of transgenic mice by pronuclear injection. Methods Enzymol. 101: Hogan, B., R. Beddington, F. Costantini and E. Lacy Manipulating the Mouse Embryo. A Laboratory Manual, 2 nd ed., pp Cold Spring Harbor Laboratory Press. NY, USA. Ju, J. C., S. P. Cheng, P. C. Tarng and K. B. Choo In vivo development and micro-injection of rabbit zygotes. Asian. J. Anim. Sci. 4: Peterson, K. R., C. H. Clegg, C. Huxley, B. M. Josephson, H. S. Haugen, T. Furukawa and G. Stamatoyannopoulos 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:

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

326 outbred ICR 2 3 g ml 2.5% Avertin 90 mm petri dish 70% A cm 1 cm B C 5 bursa D 6 M µm Pasteur pipette M2 medium M2 medium A 7 B M2 medium C 8 319

327 utero-tubal junction 5 mm 26G 6 M2 medium 200 µm medium medium

328 (A) 70% (B) 2-3 cm 1 cm (C) (D) bursa M2 medium (A) (B) (C) (A) µm M2 medium M2 medium (B) (C) 321

329 Utero-tubal junction (A) (B) (A) 26G (B)

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

331 DNA DNA genomic DNA polymerase chain reaction, PCR Southern blot hybridization DNA DNA DNA DNA DNA ml 1.5 ml UV % 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

332 70% EtOH TE buffer 5 mm Tris-HCl, ph8.0; 0.1 mm EDTA 0.8% agarose gel EtBr 10 mg/ml g 10 ml 2 ml 0.9% NaCl rpm ml Solution A 1 ml Solution B ml Solution B 40 µl proteinase K solution phenol rpm ml chloroform 1/25 isoamyl alcohol rpm ml 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 DNA 260 nm 1 50 µg/ml 325

333 DNA ml 1.5 ml UV 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 ml 10 ml rpm 10 90% 3. 7 ml M NH 4 Cl rpm % NaCl 5. 2 ml TNE buffer µl proteinase K solution C phenol 326

334 9. 3,000 rpm ml chloroform 1/25 isoamyl alcohol 10. 3,000 rpm ml 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

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

336 ---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

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

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

339 2. DNA prehybridization solution 100 µl/cm N NaOH 37 5 denature prehybridization solution 5 25 ng/ml cpm/ml prehybridization solution 10 ml/100 cm X SSPE/0.5% SDS 15 1X SSPE/0.5% SDS X SSPE/1% SDS MM X intensifying screen

340 3MM 3MM 333

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

342 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, > 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

343 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 phenol chloroform 12,000 rpm 10 DNA TE DNA DNA DNA 100 ng PCR DNA PCR PCR PCR 50 µl 1X PCR 2.5 mm MgCl 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

344 min DNA sec 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 l M NH 4 OAc, ph 4.8 DNA Slot-blot Minifold II DNA UV crosslinker 1,200 DNA NC 25 ml /4 32 P > cpm cdna NC 0.1% SDS/2X SSC % SDS/0.1X SSC % SDS/0.1X SSC NC PE X 337

345 autoradiography Slot-blot DNA 338

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

347 Chen, C. M., K. B. Choo and W. T. K. Cheng 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: Chen, C. M., C. H. Wang, S. C. Wu, C. C. Lin and W. T. K. Cheng Temporal and spatial expression of biologically active human factor VIII in the milk of transgenic mice driven by bovine -lactalbumin promoter. Transgenic Res. 11: Chen, C. M., C. L. Hu, C. H. Wang, C. M. Hung, H. K. Wu, K. B. Choo and W. T. K. Cheng Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene. Mol. Reprod. Dev. 54: Sambrook, J., E. F. Fritsch and T. Maniatis Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, USA. 340

348 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

349 0.1% DEPC 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

350 Stock solution ddh 2 O/DEPC 293 ml 0.75 M sodium citrate ph ml 10% w/v N-lauroylsarcosine Sarkosyl 26.4 ml guanidine thiocyanate 250 g 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 g sodium acetate anhydrous 40 ml 35 ml glacial acetic acid glacial acetic acid ph ml 4. Water-saturated phenol C 100 g phenol crystals 4 C 5. 49:1 v/v chloroform/isoamyl alcohol bromochloropropane % isopropanol 7. 75% ethanol DEPC ddh 2 O 8. deionized formamide mg ml denaturing solution pipetman ml 2 M sodium acetate 1 ml Water-saturated phenol 0.2 ml 49:1 v/v chloroform/isoamyl alcohol or bromochlo- ropropane C 10,000 g 20 RNA aqueous phase 1 ml DNA organic phase 4. 1 ml 343

351 1 ml isopropanol -20 C C 10,000 g 10 pipetman ml isopropanol -20 C 30 4 C 10,000 g 10 pipetman 7. 1 ml DEPC 75% C 10,000 g RNA 9. RNA ddh 2 O/DEPC formamide C RNA 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 mg ml TRIzol 344

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

353 0.5 ml isopropanol C 12,000 g 10 pipetman 6. 1 ml ddh 2 O/DEPC 75% 4 C 7,500 g RNA 7. RNA ddh 2 O/DEPC 0.5% SDS 1 mm EDTA ph C RNA C RNA RNA ddh 2 O/DEPC UV spectrophotometer 260 nm 280 nm RNA A 260 /A 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)

354 pp Chomczynski, P. and N. Sacchi Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: Gilman, M 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 Extraction, purification, and analysis of mrna from eukaryotic cells. In: Molecular Cloning, A Laboratory Manual. 3 rd ed., pp Cold Spring Harbor Laboratory Press, NY, USA. 347

355 ---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

356 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 X 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

357 % H 2 O 2 10 H 2 O 2 3. ddh 2 O/DEPC % 1 g agarose 250 ml 72 ml ddh 2 O/DEPC C ml 10X Formaldehyde gel-running buffer 18 ml (teeth comb) ml Reagent µl ddh 2 O/DEPC + RNA ( µg) 11 10X Formaldehyde running buffer M Formaldehyde 9 Formamide 25 Total volume C RNA 10 µl formaldehyde gel-loading buffer 2 µl EtBr EtBr ddh 2 O/DEPC formaldehyde gel-running buffer 5 V/cm RNA 5 V/cm bromophenol blue BPB 2/3 UV transilluminator box RNA 350

358 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 M NaCl ddh 2 O/DEPC 1. ddh 2 O/DEPC ml 20X SSC 5 3. RNA X SSC 2 351

359 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 C 352

360 2. DNA 0.2 M EDTA, ph M LiCl Random priming DIG Kit Boehringer Mannheim; Roche 100% ethanol TE buffer 10 mm Tris-HCl, ph 8.0; 1 mm EDTA ng DNAddH 2 O 15 µl µl hexanucleotide mixture + 2 µl dntp mixture + 1 µl Klenow enzyme 37 C 2 42 µl 0.2 M EDTA DNA 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

361 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 ml Reagents Volume (L Final concentration ddh 2 O PCR buffer PCR DIG mix µm dntp T3 promoter primer µm T7 promoter primer µm pblusgus DNA 1 ~50 pg Taq DNA polymerase U Total volume 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

362 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 ml 6X SSC 5 3. Whatman 3MM

363 4. Crosslinker UV ml hybridization buffer 42 C prehybridization C ml 2X SSC 0.1% SDS 5 Geiger counter ml 0.2X SSC 0.1% SDS 42 C ml 0.1X SSC 0.1% SDS 50 C 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

364 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 ml Washing solution ml Soultion µl anti-dig-ap-ab 10 ml Soultion 2 50 rpm ml Soultion 2 15 Ab ml Soultion ml color-substrate solution ml Soultion 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 µm cdna

365 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 mm dntp datp, dttp, dctp, dgtp 200 mm Sodium pyrophospate Primers oligo(dt) 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 µl 10X RT buffer 3 pmol 1 2 µl ddh 2 O/DEPC 25 µl 4. oligo(dt) 4 C 15 random hexamer Reagent µl 10 RT buffer 2.5 BSA 5 µg/µl 1 RNasin 5 U/µL mm β-mecaptoethanol mm datp mm dttp mm dctp mm dgtp mm Sodium pyrophospate 1 ddh 2 O/DEPC 14.5 Total volume RNAs/primers 25 µl reverse transcriptase 1 µl

366 7. 42 C 45 cdna single strain complementary DNA C PCR ThermoScript TM RT-PCR System ThermoScript TM RT-PCR System Invitrogen, Life Technologies Reagent µl ThermoScript TM RT 15 U/µL cdna synthesis buffer M Dithiothreitol DTT 250 RNaseOUT TM 40 U/µL mm dntp Mix Oligo (dt) 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 M DTT 1 10 mm dntp Mix 2 DEPC-Treated Water X Total volume C 5 RNA µl cdna synthesis mix Reagent µl ThermoScript TM RT 15 U/µL 1 5 cdna synthesis buffer M DTT 1 RNaseOUT TM 40 U/µL 1 DEPC-Treated Water 1 5. oligo(dt) C 359

367 30 60 random hexamer C cdna C µl RNase H 37 C 20 RNA C 2 µl PCR 1. DEPC formaldehyde formamide ethidium bromide 2. UV 360

368 pp Brown, T 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 cdna cloning by RT-PCR. In: PCR 2-A Practical Approach. pp IRL Press, NY, USA. Sambrook, J. and D.W. Russell Extraction, Purification, and Analysis of mrna from Eukaryotic Cells. In: Molecular Cloning: A Laboratory Manual. 3 rd ed., pp Cold Spring Harbor Laboratory Press, NY, USA.. 361

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

370 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 ml/ 0.5 ml 20 µl 80 µl 10 mg Micrococcus lysodeikticus 3 ml 0.1 M phosphate buffer, ph ml 20 µl 180 µl microplate nm O.D ph nm A 450 = bovine serum albumin, BSA BCA 1 reagent B 50 reagent A Working reagent WR25 µl BSA microplate 200 µl WR nm O.D. 363

371 reducing 12% SDS-PAGE 7.mm 8 mm 50 µl sample buffer 50 µl 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

372 365

373 2002 : Ford, S. R Biochemistry 2344 Lecture 10: Protein Purification. ures/lecture%2010--enzyme%20purification/2344%2flectur e%2010-lysozyme%20purif Millard, J. T Experiment 3: Purification and characterization of the enzyme lysozyme. 3.pdf Laemmli, U Cleavage of structural protein during the assembly of the head of Baxcteriophage T4. Nature 227: Pierce com BCA Proein Assay Kit

374 ---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

375 SDS-PAGE 1. homogenate buffer 1 10 w/v 4 10,000 g 10 supernatant Bradford 1976 BSA µg SDS-PAGE sample buffer 1 1 Laemmli 1970 SDS-PAGE 5% stacking gel % separating gel gradient V 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 hybridization and detection 1. TBS 2 368

376 5% skim milk TTBS blocking solution 1 blocking Blocking solution 3% gelatin bovine serum albumin 2. blocking 1% TTBS 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 ECL Amersham RPN 2106 alkaline phosphatase conjugated CSPD A B 1 2 X Biomax; Kardo

377 3MM 3MM 370

378 Bers, G. and D. Garfin Protein and nucleic acid blotting and immunochemical detection. Biotechniques 3: Bradford, M. M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ana. Biochem. 72: Hong, D. H., J. Huan, B. R. Ou, J. Y. Yeh, T. C. Saido, P. R. Cheeke and N. E. Forsberg Protein kinase C isoforms in muscle cells and their regulation by phorbol ester and calpain. Biochim. Biophys. Acta 1267: Towbin, H., T. Staehelin and J. Gordon Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: Yeh, J. Y., S. C. Vendeland, Q. P. Gu, J. A. Butler, B. R. Ou and P. D. Whanger Dietary selenium increases selenoprotein W levels in rat tissues. J. Nutr.. 127:

379 Dept. of Developmental Biology, University of Cincinnati, USA, Ph.D. Children's Hospital Medical Center, Cincinnati, USA, PDF Assistant research Fellow, IMB, Academia Sinica, Taipei present Associate research Fellow, IMB, Academia Sinica, Taipei PURDUE TUFTS

380 ( ) [email protected] [email protected] [email protected] [email protected] (1995-present) [email protected] [email protected] ~ [email protected] Ellis Fischel [email protected] 373

381 The Sanger Institute, Wellcome Trust, Cambridge, U.K

382 Dept. of Developmental Biology, University of Cincinnati, USA, Ph.D Davis

383 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 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

384 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,

385 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 Immunoglobulin 214,257 Ligand 251 In and out Line spectrum In vitro Lipofection 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 Incident Luciferase Individual ventilated cage IVC 268 Luciferin 133 Influenza virus Lytic cycle 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, Insertional mutation im 265,276 Mastitis 215 Insertional recombination 189 Matrix 251 Insulin-like growth factor IGF-1 Maturation promotion factor MPF ,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, Micro-isolator 268 Interphase 20 Micropyl 145 Intrachromosomal recombination Milk protein binding factor MPBF Integration 58 Mobile elements 265 Intron 161,203 Monomer 133 Inverted repeats 74,163,193 Morula 292,318 Isoelectric point pi 249, Isogenic 157 J Jellyfish 131 K Knock in 163 L Lactase 214 Lactate 45,290 Lactoferrin LF 214,248 Lactose 213 Lampbrush chromosome 19

386 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 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

387 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, 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