Nature (2003) 422:

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1 The Studies and Applications of Transgenic and Cloned Animals 1

2 Nature (2003) 422:

3 James Watson Francis Crick 1953 DNA (double helix) 1962 Watson and Crick / Genome = Gene + Chromosome 3

Nature (2003) 422:835-847 The future of genomics

4 Nature (2003) 422: The future of genomics rests on the foundation of the Human Genomic Project 4

5 DNA 5

6 6

7 DNA 7

8 (transgenic animal) (genome) DNA 1. (microinjection) 2. (retroviral vector infection) 3. (sperm vector) 4. (embryonic stem cell) 5. (nuclear transplantation) 8

9 - - α- β- ( 1999) 9

10 1968 Gardner et al., Nature 220, Brackett et al, PNAS 68, 353 DNA Jaenisch et al., Cold Spring Harb. Symp. 39, 375 Jaenisch et al., PNAS 72, 4008 Gordon et al., PNAS 77, 7380 Gordon et al., Science 214,1244 Brinster et al., Cell 27, 223 Palmiter et al., Nature 300, 611 Campbell et al., Nucleic Acids Res. 12,

1993 1995 1996 1997 C Velander et al., PNAS 89, 12003 - enzymatic Drews et al., PNAS 92, 10462 modification Schellander et al., Nat. Biotechnol. 6, 41 Campbell et al., Nature 380, 64 Thomson et al.

11 C Velander et al., PNAS 89, enzymatic Drews et al., PNAS 92, modification Schellander et al., Nat. Biotechnol. 6, 41 Campbell et al., Nature 380, 64 Thomson et al., Biol. Reprod. 55, 254 ES Wilmut et al., Nature 385, 810 Schnieke et al., Science 278, 2130 Paleyanda et al., Nat. Biotechnol. 15, 971 superoxide Stromqvist et al., Transgenic dimutase Res. 6,271 Dolly Petters et al., Nat. Biotechnol. 15,

12 Loi et al., Nat. Biotechnol. 19, 962 Wakamatsu et al., PNAS 98, 1071 Shin et al., Nature 418, 732 Chesne et al., Nat. Biotechnol. 20, 366 Lee et al. Nature Biotechnol. 20: Galli et al., Nature 424, 635 Woods et al., Science 301, 1063 Zhou et al., Science 302, Kono et al. Nature 428: ( ) 12

13 Development of Vertebrate Embryos

14 14

15 1st Polar body Meiotic spindle 1st Polar body 15

16 Stereo microscope 1~100X ( ) 16

17 , (transgene) : DNA (promoter), polyadenylation site, complete coding region, at least one intron, Linearizaton of the cloned DNA, The removal of plasmid vector sequences µl of DNA (1 to 5 µg/ml) (transgenic animal) PCR, Southern blot, Western blot, Immunohistochemistry. 17

18 ( / ) -4.9% -6.9% -8.39% -9.2% % DNA ( 1999) 18

19 19

20 40~1,000 X

21 Foreign Gene Microinjection Female pronucleus Male pronucleus Palmiter et al. (1982) / Science 385:

22 (Puller) (Grinder) (Microforge) (Injector) 22

23 (injection pipette) (hair) 23

24 24

25 40~1,000 X

26 (retroviral vector infection) Wheeler & Walters (2001) Theriogenology 56:

27 Intracytoplasmic sperm injection (ICSI) (sperm vector) Injection Holding Pipette 27

28 (embryonic stem cell) Donovan and Gearhart (2001) Nature 414,

29 / (electroporation) DNA ( 2004) Before During After Electron micrographs of cells before and after brief electric pulses confirm that electroporation causes pores to open and close. 29

30 / (Equipments for Electroporation/ fusion) 30

31 (gene/particle gun) DNA 31

32 Cloning of Embryos 32

33 33

34 /SvJ+C57BL/6 34

35 Chimeras Zebhund oder Seera Kängaffe oder Girruh 35

36 Techniques of nuclear transfer in mammals Keith et al. (1996) / Reviews of Reproduction 1:

37 (Enucleation) (Nuclear Transplantation) PB1 Donor cell A C PB1 Donor cell ( 2004) B D 37

38 (Electrofusion) A B C D 50 m Procedures of nuclear transfer in cattle ( 2004) 38

39 Cloning of Sheep Dolly and her mother Mammary cells 1/277 = 0.4% Nuclear transplantation Wilmut et al. (1997) / Nature 385:

40 Cloning of Sheep Megan and Morag first animals cloned from cultured cells Dolly and her 3 lambs born in April

41 Frozen Matured oocyte Embryo culture Culture and passages Enucleation Donor cells Starvation Nuclear transfer Procedure of cattle cloning Electrofusion and Activation ( 2004) 41

42 (Cloning of Transgenic Calves) ACT3, ACT4, ACT5; 3 weeks of age A,B. Transgenic fibroblast C,D. Nontransgenic fibroblast N C A D Normal PCR amplified segment of the pcmv/βgeo construct of DNA from cell line and calf Cibelli et al. (1998) Science 280: /276 =1.8% Southern blot of genomic DNA 42

43 Cloning of Mouse Wakayama et al., (1998) Nature 394: /1385 = 2.2% No. Oocyte

44 Cloning of Pig Single NT 0/185 = 0% - 3 Recipients No. oocytes Double NT 5/401 = 1.3% - 7 Recipients No. oocytes Polejaeva et al. (2000) Nature 407:

45 Bisection of embryos CLONING PROCEDURE Chimeras with ES cells Nuclear transfer 2-cell embryos Blastocyst ES cells Blastomeres Cultured cells Injected into host embryos Transferred into enucleated oocyte Production of bisected blastocyst Transferred into recipient Slection and mating of germ-line chimeras Transfer into recipient Transferred into recipient Cloned pigs ( 2004) 45

Applications of transgenic and cloning techniques Transgenic animal technology

cells Research models Animals as bioreactor Disease-resistant animals Improve

targetting) Gender and genetic consistent Clone elite animals Endangered breed

46 Applications of transgenic and cloning techniques Transgenic animal technology Nuclear transfer technology Transgenic animals Animal cloning Embryonic stem cells Research models Animals as bioreactor Disease-resistant animals Improve production performance Xenotransplanation Transgenic cloning Animal models (gene targetting) Gender and genetic consistent Clone elite animals Endangered breed Xenotransplanation Provide endogenic cells Therapeutic cloning Drug screening ( 2004) 46

Cloned female mouflon with the sheep foster

47 Cloned female mouflon with the sheep foster mother Nuclear source: Granulosa cells from dead ones 1/22 = 4.5% Loi et al. (2002) Nature Biotechnology 19: a. Nuclear-donor (cumulus cells) cat; b. cloned kitten with its surrogate mother. 1/87 = 1.2% Shin et al. (2002) Nature 415:859 47

48 Rabbits born from somatic nuclear transfer Nuclear source: Cumulus cells 6/755 = 0.8% (2 died) A1 EGFP protein Chesne et al. (2002) Nature Biotechnology 20:

49 Mouse created without father Kaguya 2/371 = 0.6 % (survived) No. Oocytes Parthenogenesis ( ) Kono et al. (2004) Nature 428: Imprinted genes ( ) 49

50 0.75 h 1 h 1.25 h 1.5 h 1.75 h 2 h 10 min 2.5 h 3.3 h 3.5 h 40 min Bar = 250 µm 3.8 h 4.3 h 4.7 h 50

51 24 h 28 h 28 h 3.3 h 3.3 h 36 h 36 h 42 h 42 h 120 h 48h 48 h 60 h 60 h 72 h 72 h 120 h 51

52 Saga and Takeda (2002) Nature Reviews Genetics 2:

53 53

54 a b a. ( ) b. ( 30 ) c c. 54

55 55

56 ( ) 56

The Construction of Expression Systems PCR 1 ACCATGGCTC TCTCACTTTT CACTGTCGGA CAATTGATTT CTTATTTTG 51 GACAATGAGA ATCACTGAAG CCAACCCCGA CCCCGCAGCC AAGCCGTCC 101 CTGCCGCAGC CGCCCCAGAC ACCGCCTCTG

57 The Construction of Expression Systems PCR 1 ACCATGGCTC TCTCACTTTT CACTGTCGGA CAATTGATTT CTTATTTTG 51 GACAATGAGA ATCACTGAAG CCAACCCCGA CCCCGCAGCC AAGCCGTCC 101 CTGCCGCAGC CGCCCCAGAC ACCGCCTCTG ATGCCGCCGC GCAGCCGCC 151 GCCACCGCAG CCACCGCCGC CGCAGCAGCC GCCGCCACCG AGTCACCGC 201 TGCCAAAGCC GCAGCCCTCA CCGCCGCCAA CGCCGCCGCC CCGCAGCAG 251 CCACCGCCGC CGCAGCCGCC AGAGGTTAA (Vector) (Reporter gene) 57

58 DNA A B 58

59 1% agarose 59

60 A B C1 C2 60

61 EGFP-N1 (Vector alone) a1 b1 c1 a2 b2 c2 61

62 62

63 28 h 84 h 36 h 108 h 48 h 120 h 60 h 144 h 72 h 168 h 63

64 Ju et al. (1999) Devel. Genetics 25:

65 (gene knockdown) Morpholino or RNAi (translation) Nature Genetics (2000) 26:

66 Nuclear Transplantation in Fish Medaka Donor Wild type Recipient Orange red 29/845 = 3.4% D R F1 MD FD FR Niwa et al. (1999) / Develop Growth Differ 41:

67 Cloned Zebrafish ~ 2% Lee et al. (2002) / Nature Biotechnol. 20:

68 vs Devlin et al. (2001) Nature 409,

69 (a) (b) (c) (d) 69

70 Thank you! Vielen Dank! Merci! 70

兩願離婚之研究

兩願離婚之研究裂不 1 女女不 sol-sol-sol-mi 令不女不念力沈異諾不來倫理律兩 Paganini 靈不來了靈羅靈 1 年 62 1 2 La campanella 律若說 3 不理 2 說女律理不不了不類律類理歷 3 1860 年 chromosome 葉 1944 年 DNA 良了類 2 1 40 3 理錄諾年 4