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Animal Copy

  1. 1. 動物複製之產製技術與應用 沈朋志 國立屏東科技大學 動物科學與畜產系
  2. 2. 大 綱  核轉置家畜之產製流程  供核細胞的製備  受核卵母細胞之去核操作  細胞電融合操作  核轉置胚之激活處理  轉基因複製牛羊之產製
  3. 3. Fig. Nuclear transplantation procedure in rabbit. (Stice and Robl, 1988) 2N 2N
  4. 4. 複 製 牛 羊 之 產 製 流 程 Ear cells Culture and passages Donor cells Matured oocyte Enucleation Nuclear transfer Electrofusion and Activation Embryo culture and transfer
  5. 5. C D A B Fig. Procedures of nuclear transpfer in cattle. Bar=50 μ m.
  6. 6. 受核卵母細胞之去核操作 Collection of goat recipient oocytes Superovulation 22 ~ 24 h after hCG injection Remove cumulus NT
  7. 7. Collection of bovine recipient oocytes COCs collection IVM Remove cumulus
  8. 8. 1. GVBD 期之染色體位於卵母細胞之中央位置。 2. MⅡ 期之染色體位於細胞表面,並排出第一極體。 3. MⅡ 期卵母細胞之染色體位於第一極體之相對應端。 4. 因此,去核操作時即以第一極體為基準,供為判定染色體之位置。 卵母細胞之染色體於成熟過程中之位置變化
  9. 9. 卵母細胞去核成功與否之判定 ( 利用 DNA 染劑 ( 如 :Hoechst 33342) 辨識染色體位置 ) 去核前染色 ( 紫外燈照射之時間長度應控制在 30 秒以內 ) 去核後染色 ( 染移除之細胞質部分不需擔心 紫外燈照射之時距 )
  10. 10. Fig. Diagram of oocyte showing 4 positions in relation to the first polar body. Ⅰ Ⅱ Ⅲ Ⅳ
  11. 11. Fig. Enucleation Confirmation of micromanipulated bovine oocytes by Hoechst 33342 staining. A’ B A B’
  12. 12. ( 沈等 , 2000)
  13. 13. ( 沈等 , 2000)
  14. 14. 不同取卵時間及第一極體形態對小鼠卵母細胞內染色體位置之影嚮 Collection PB1 mor- No. of No.(%) of oocyte with chromosome position, in oocytes time, (hr)1) phology examined Ⅰ Ⅱ Ⅲ 10 ~ 11 intact 100 82(82.0) a 13(13.0) 5( 5.0) degenerated 89 43(48.3) b 39(43.8) 7( 7.9) total 189 125(66.1) A 52(27.5) 12( 6.4) 12 ~ 13 intact 221 119(53.8) b 74(33.4) 28(12.7) degenerated 213 77(36.2) c 105(49.3) 31(14.5) total 434 196(45.2) B 179(41.2) 59(13.6) 14 ~ 15 intact 35 15(42.9) b 15(42.9) 5(14.2) degenerated 185 58(31.4) c 76(41.1) 51(27.5) total 220 73(33.2) C 91(41.3) 56(25.5) 1) Time =hours after hCG injection. ( 沈 , 1997) a,b,c or A,B,C Within columns, values with different superscripts differ( p<0.05). 29
  15. 15. 不同取卵時間及第一極體形態對家兔卵母細胞內染色體位置之影嚮 Collection PB1 No. of No.(%) of oocyte with oocytes chromosome position, in time, (hr) 1) morphology examined Ⅰ Ⅱ Ⅲ 12 ~ 13 intact 57 53( 93.0) 4( 7.0) 0 degenerated 1 1(100.0) 0 0 total 58 54( 93.1) 4( 6.9) 0 14 ~ 15 intact 46 41( 89.1) 5(10.9) 0 degenerated 24 20( 83.3) 4(16.7) 0 total 70 61( 87.1) 9(12.9) 0 16 ~ 17 intact 44 42( 95.5) 2( 4.5) 0 degenerated 17 15( 88.2) 2(11.8) 0 total 61 57( 93.4) 4( 6.6) 0 1) Time =hours after hCG injection. ( 沈 , 1997) 30
  16. 16. 細胞電融合
  17. 17. 電 穿孔 / 融合 之設備 (Equipments for Electroporation/ fusion)
  18. 18. Electron micrographs of cells before and after brief electric pulses confirm that electroporation causes pores to open and close. Before During After 細胞電融合操作
  19. 19. 電激法引導細胞融合之小電孔 紅血球經電激後表膜將形成兩種口徑之電孔,大者達 8.3 nm 以上,而小者僅約 0.5 nm ;於電激處理後之 100-200 毫秒 (ms) 內,已形成之大口徑電孔又隨即癒合,而小口徑電孔則仍維持於開放狀態。
  20. 20. Fig. Macroscopically observable events as they rigorously relate to functional membrane fusion (Sowers, 1988) 33
  21. 21. 影響細胞電融合效率之因子 : 1. 擬融合細胞之排列方向和細胞間之接觸緊密性 2. 電融合之電參數組合 3. 供核細胞之細胞週期期別 4. 受核卵母細胞之年齡
  22. 22. 1. 擬融合細胞之排列方向和細胞間之接觸緊密性 A. 欲控制被融合細胞與白金絲之排列方位,可經由交流電或人為之方法執行 B. 供受核兩細胞間接觸之界面需與融合槽之兩白金絲平行 ( 即電場方向垂直通過兩細胞接觸之界面 ) C. 細胞質移除過多,則卵膜間隙之空間將相對擴大,當所置入之供核胚葉細胞之體積過小時,則受核與供核細胞間之接觸性不緊密,在電激前經由交流電之聚集雖可暫時性令透明帶內之兩細胞緊密接觸,惟在交流電操作後,兩細胞間之接觸狀態,極易因電融合操作或核移置胚之移動性吸放操作再度分離,令融合率下降
  23. 23. 2. 電融合之電參數組合 A. 電場強度 B. 電激持續時間 C. 電激次數
  24. 24. Parameters for electrical fusion in mammalian embryos (Robl et al .,1992)
  25. 25. 表 .電融合之電場強度對完整二細胞期兔胚二胚葉細胞之 融合率與融合胚於體外後續發育之影響 Pulse No. of No. (%) of No. (%) of fused embryo developed to strengths 1) embryos 2) embryos (kv/cm) treated fused Uncleavage 16-cell M + B 3) 2.0 32 22(68.8) a 1( 4.5) 19(86.4) 18(81.8) 2.5 56 55(98.2) b 3( 5.4) 49(89.0) 45(81.8) 3.0 30 29(96.7) b 1( 3.4) 26(89.7) 25(86.2) 3.5 30 26(86.7) ab 4(15.4) 21(80.8) 17(65.4) control 66 0 61(92.4) 48(72.7) 1) Electrofusion parameter: Different field strengths + 60μs + 2 pulses. 2) Two- cell stage embryos were collected just at 1-2 hours postcleavage. 3) M+B : morula or blastocyst. a-b Data within columns,values with different superscripts differ(p<0.01)
  26. 26. 表 . 電融合之電激次數對完整二細胞期兔胚二胚葉細胞 之融合率和融合胚於體外後續發育之影響 Pulse No. of No. (%) of No. (%) of fused embryo developed to embryos fused number treated 1) embryos 2) Uncleavage 16-cell M+B 3) 1 36 31(86.1) 1( 3.2) 28(90.3) 23(74.2) 2 31 27(87.1) 4(14.8) 22(81.5) 18(66.7) 1) Two- cell stage embryos were collected just at 1-2 hours post-cleavage. 2) Electrofusion parameter: Different pulse numbers + 2.5 kv/cm + 60μs. 3) M+B : morula or blastocyst.
  27. 27. Table . In vitro development of caprine NT-embryos cloned with adult ear fibroblast cells after activation with different electrical field strengths Filed strength No. (%) of NT-embryos produced No. (%) of NT-embryos fused No. of NT-embryos cultured No. (%) of NT-embryos developed to 2-cell after culture 1.67 kV/cm 2.33 kV/cm 93 108 60 (64.5) 70 (64.8) 51 61 10 (19.6) a 40 (65.6) b a, b Values within columns with different superscripts differ significantly (p<0.01).
  28. 28. Table . The results of embryo transfer with caprine NT- embryos cloned from adult ear fibroblast cells after activation with different electrical field strengths     Filed strength 1.67 kV/cm 2.33 kV/cm No of NT- embryos transferred   51 61 No. of Recipients   14 7 No. (%) of recipients became pregnant   0 ( 0.0) 2 (28.6) No. (%) of born   0 (0.0) 3 (4.9)
  29. 29. 3. 供核細胞之細胞週期期別 供核細胞之細胞週期為早期之小鼠二細胞期胚葉細胞 ( 自單細胞期分裂為二細胞後 1.5 hr) 為供核時,其核移置胚之融合率明顯較以細胞週期為中期 ( 自單細胞期分裂為二細胞後 5-6 hr) 或晚期 (hCG 注射後 48 hr) 者為低。
  30. 30. 4. 受核卵母細胞之年齡 體外成熟培養 24 hr 之牛卵母細胞所產製之核移置胚之細胞融合率,較利用經體外培養 42 hr 者為佳
  31. 31. A schematic drawing of the possible mechanism of cell signalling in the fertilized hamster egg. (Mayazaki, 1990) 核轉置胚之激活處理
  32. 32. Table . Effects of activation agents or chemicals in oocyte activation ( 劉 , 2000)
  33. 33. Table . I n vitro development of bovine NT-embryos activated by electrical pulse or calcium ionophore combined with 6-DMAP at 24 h after IVM Activation agents * E A23187 No. of fused NT- embryos 59 64 No. (%) of development ☆ 2-cell 16-cell CM B 51 (86.4) 28 (47.5) 27 (45.8) 18 (30.5) 59 (92.2) 38 (59.4) 36 (56.3) 27 (42.2) * E: Oocytes activated by an electrical pulse combined with 6-DMAP; A23187: Oocytes activated by calcium ionophore combined with 6-DMAP. ☆ CM: Compacted morula; B: blastocyst. ( 沈等 , 2003)
  34. 34. ( 沈等 , 2003) Table . T he in vitro development of bovine NT-embryos activated by calcium ionophore (A23187) or ionomycin combined with 6-DMAP at 24 h after IVM Activation agents * A23187 I No. of fused NT- embryos 90 93 No. (%) of development ☆ 2-cell 16-cell CM B 76 (84.4) 42 (46.7) 41 (45.6) 33 (36.7) 81 (87.1) 41 (44.1) 38 (40.9) 31 (33.3) * A23187: Oocytes activated by calcium ionophore combined with 6-DMAP; I: Oocytes activated by Ionomycin combined with 6-DMAP. ☆ CM: Compacted morula; B: blastocyst.
  35. 35. ( 沈等 , 2003) Table . The in vitro development of bovine NT-embryos activated by calcium ionophore (A23187) combined with different activation stimuli Activation agents * 6-DMAP CHX + CB-1 CHX+ CB-5 No. of fused NT- embryos 95 49 49 No. (%) of development ☆ 2-cell 16-cell C M B 94 (98.9) a 57 (60.0) a 53 (55.8) a 49 (51.6) a 26 (53.1) c 9 (18.4) c 6 (12.2) b 4 ( 8.2) b 39 (79.6) b 17 (34.7) b 9 (18.4) b 7 (14.3) b * 6-DMAP: Oocytes were activated by A23187+ 6-DMAP (4 h); CHX+CB-1: Oocytes were activated by A23187 combined with cycloheximide and cytochalasin B (1 h) and then with cycloheximide alone for 4 h; CHX +CB-5: Oocytes were activated by A23187+cycloheximide and cytochalasin B (5h) . a, b Values with different superscripts in the same column differ (p<0.01). ☆ CM: Compacted morula; B: blastocyst.
  36. 36.  The interaction of cell cycle between recipient oocyte and donor cell 48
  37. 37. M G2 S G1 G0 Growth factor absent Growth factor present Cell cycle stage 2N 2~4N 4N 4N 49
  38. 38. Table Development in uitro of nuclear transfer embryos derived from non-starved or starved bovine fetal fibroblasts Number of aryoplast- Fused Cleaved* Blastocyst* Hatched Fibroblast type cytoplast complexes (%) (%) (%) blastocyst*(%) Non-starved 242 174(72) a 115(66) a 35(20) a 24(14) a Starved 254 205(81) a 158(77) b 80(39) b 58(28) b Total 496 379(76) 273(72) 115(30) 82(22) *Percentages were calculated on the basis of the number of fused embryos. Ab Values within columns with different superscripts differ(P<0.05). (Zakhartchenko et al ., 1999) 54
  39. 39. Table . Effects of activation strategies on t he in vitro development of bovine NT-embryos cloned with adult ear fibroblast cells Activation strategy * FAS FBA No. of fused NT- embryos 39 42 No. (%) of development ☆ 2-cell 16-cell CM B 28 (71.8) 6 (15.4) b 6 (15.4) b 2 ( 5.1) b 37 (88.1) 19 (45.2) a 18 (42.9) a 11 (26.2) a * FAS: Fusion and activation simultaneously; FBA: Fusion before activation. a, b Values with different superscripts in the same column differ significantly (p<0.01). ☆ CM: Compacted morula; B: blastocyst. 56 ( 沈等 , 2003)
  40. 40. Table The blastocyst rate of somatic cell nuclear transfer embryos derived from 3 types of bovine cells 供核細胞種類 (Modified from Cho et al., 2004) 46(15.2) e) 203(67.0) d) 303(79.9) d) 379 Ear fibroblasts 96(28.7) d) 245(73.4) d) 334(78.4) d) 426 Cumulus cells Development to blastocysts (%) b) Cleaved (%) b) Fused (%) a) Injected 15(7.0) c) 109(50.9) c) 214(59.0) c) 363 Fetal fibroblasts No. of embryos Types of donor cells Model effect of the donor cell on the number of embryos fused, cleaved, developed to the blastocysts and expressed, which was indicated as a P value, was 0.0001, 0.0001, 0.0001 and 0.0049, respectively. a) Percentage of the number of embryos injected. b) Percentage of the number of embryos fused. c,d) Within a parameter, values with different superscripts differed significantly P<0.05.
  41. 41. Table Development rates of transgenic somatic cell nuclear transfer embryos reconstructed with cumulus cells at different cell passages Model effect of the subculture after transfection on the number of embryos fused, cleaved, developed to the blastocysts and expressed, which was indicated as a P value, was 0.1798, 0.3106, 0.8650 and 0.7088, respectively. a) Percentage of the number of embryos injected. b) Percentage of the number of embryos fused. (Modified from Cho et al., 2004) 33(17.9) 129(70.1) 184(66.7) d) 276 Late (8-12 passage) 29(14.4) 138(68.3) 202(76.5) c) 264 Early (2-4 passage) Development to blastocysts (%) b) Cleaved (%) b) Fused (%) a) Injected No. of embryos Cell passages
  42. 42. Table Comparison of development rates of bovine transgenic somatic cell nuclear transfer embryos derived from different size of transfected cumulus cells Model effect of the size on the number of embryos fused, cleaved, developed to the blastocysts and expressed, which was indicated as a P value, was 0.9678, 0.4093, 0.0340 and 0.8835, respectively. a) Percentage of the number of embryos injected. b) Percentage of the number of embryos fused. c,d) Within a parameter, values with different superscripts differed significantly, P<0.05. (Modified from Cho et al., 2004) 21(23.1) d) 72(79.1) 91(75.8) 114 Large ( ≧ 30 μm ) 36(38.7) c) 78(83.9) 93(75.6) 123 Small (< 30 μm ) Development to blastocysts (%) b) Cleaved (%) b) Fused (%) a) Injected No. of embryos Size of donor cells
  43. 43. (Hayes et al., 2005) Table In vitro development to blastocysts upon nuclear transfer of cloned bovine embryos reconstructed from adult fibroblast cells subjected to serum starvation or confluency Different letters (a and b) indicate significant differences. Confluent: 10% FCS DMEM. Serum stravation: 0.5% FCS DMEM. 62 (11.99) a 243 (73.88) b 63.6 a 517 Serum starved for 3 days 93 (11.69) a 488 (51.69) a 54.6 a 795 Confluent Blastocyts (%) Cleaved (%) Cloned embryos Fusion rate (%) Reconstructed couplets Culture condition
  44. 44. (Melican et al ., 2005) Table Effect of donor karyoplast harvest method on caprine NT efficiencies 0 0 1 a (2) 269/42 151 b (44) 577/340 b (59) Complete 5(0.8) 5(5) 6 a (6) 633/96 385 a (53) 1069/726 a (68) Partial Term Day 50 # Offspring (% embryo) #Pregnancies (%) # Embryos/# recipients # Cleaved (% cleavage) # Couplets/# fused (% fusion) Trypsinization a,b Within columns, proportions with different superscripts are different (P < 0.05).
  45. 45. Producing transgenic animals by nuclear transplantation
  46. 46. 供核細胞的製備 1. 卵丘細胞 COC Cumulus cells Culture and passages Starvation Frozen
  47. 47. Fig. 14. Morphology of nuclear donor cells. (A) Cumulus cells of Holsetin cattle. (B) cumulus cells of Native yellow cattle, Bar=50 μ m. A B
  48. 48. 2. 耳朵纖維母細胞 Ear cells Primary culture Starvation Frozen 7
  49. 49. 牛耳朵細胞之初代培養
  50. 50. Fig. Morphology of ear fibroblast cells derived from an adult Holstein cow. Bar=15 μ m. 8
  51. 51. Zona pellucida 80 A B C Polar bodies Pronuclei
  52. 52. (Wall, 1996) Table Microinjection method. Examples of embryo survival and transgene integration efficiencies from several laboratories
  53. 53. Table. Comparison of the production of transgenic sheep by nuclear or pronuclear microinjection (Schnieke et al ., 1997)
  54. 54. Fig. Genetic modification of from animals by DNA microinjection vs. nuclear transfer using transfected donor cells. (Wolf et al., 2000)
  55. 55. Gene transfection and selection Generation the transgene-cloned animals 81 Ear cells Culture and passages Starvation Frozen
  56. 57. 3kb 5kb GFP M 1 2 3 4 3: Ase I + EcoO1091- 回收 M : Marker 1: uncutted 2: Ase I + EcoO1091 4: Ase I 回收
  57. 58. 電融合器 - 基因轉染器 電激槽
  58. 59. Fig. Morphology of bovine ear fibroblast cells after G-418 medium screening. A: Cells without foreign gene transfection; B: Cells transfected with foreign gene. Bar = 50μm. 82 A B
  59. 60. Fig. Expression of GFP gene in ear fibroblast cells after G-418 screening. Arrows indicated cells without GFP expression. Bar = 50μm. 84 A B C
  60. 61. a. b Values within columns with different superscripts differ significantly ( P < 0.01) 295 (63.6) b 464 Passage at 50~60% cells proliferated 73 (11.5) a 633 Passage at 100% cells proliferated 57 (10.5) a 546 No passage No.(%) of cells expressed GFP No. of cells examined Selection patterns 表 4. 選殖模式對經轉染 GFP 基因牛耳朵纖維母細胞 GFP 表現率之影響
  61. 62. a,b,c,d,e,f Values without the same superscripts in the same columns are significant difference ( P <0.05) . 18 ( 4.0) a 72 ( 5.9) a 99 (24.3) b 205 (32.9) c 198 (40.2) d 293 (56.7) e 394 (65.8) f 316 (63.2) f 449 1222 407 623 493 517 595 500 0 1 2 3 4 5 6 7 No.(%) of cells expressing GFP No. of cells examined Passeage no. after G418 selected 表 5. 轉染 GFP 基因之牛耳朵纖維母細胞經不同繼代代次篩選後之基因表現率
  62. 63. 1. 週邊效應現象 (bystander effect) <ul><li>轉基因成功之細胞將抗抗生素基因之表現產物,經由細胞間之直接接觸,或藉由該產物分泌至培養液之方式,轉而提供鄰近非轉基因細胞來對抗抗生素之毒殺作用,導致存活細胞非全然為轉基因細胞之現象 。 </li></ul>(Chen et al ., 2002)
  63. 64. 造成週邊效應之可能路徑 (1). 接觸性傳遞: 細胞與細胞直接接觸,經由細胞表面之分子來進行傳遞,傳遞物質為分子量較低之物質,如離子、代謝物 (metabolites) 、 second messengers 等 (Crocker and Feizi, 1996) 。 (2). 非接觸性傳遞: 轉基因細胞將物質分泌至培養液,而非轉基因細胞經由培養液中得到,傳遞之物質除上述分子量低的物質之外,還可傳遞分子兩較高之物質,如酵素、免疫球蛋白、轉錄因子、 DNA 、 RNA 等 (Nataliya et al ., 2005) 。
  64. 65. *Control:DMEM+10%FCS, 5-Azad:DMEM+10% FCS+0.3%μΜ 5-Azad, TSA:DMEM+10% FCS+0.3μΜ TSA,5-Azad+TSA:DMEM+10% FCS+0.3μΜ 5-Azad+0.3μΜ TSA. ☆ Up to 400 cells were counted and examined in each sample. a,b,c,d Values with different superscripts differ significantly( p < 0.05). ( 沈 , 2003) 36.6 ab 41.3 d 57.2 d 5-Azad + TSA 36.6 abc 41.3 b 37.1 abc TSA 38.9 bc 42.8 c 36.3 ab 5-Azad --- --- 33.2 a Control 7 5 3 Drugs* --------------------- days --------------------------------- GFP gene expression frequency (%) after cultured for ☆ Table Effects of 5-Azad/TSA and culture periods on GFP expression in the bovine ear fibroblast cell of which transfected with GFP gene
  65. 66. Fig. Donor cells with GFP expression. A: Cells under bright light; B: Cells under FITC filters. Bar = 50 μ m. 76 A B
  66. 67. Table . In vitro development of bovine NT-embryos cloned with GFP gene transfected adult ear fibroblast cells Status of donor cell No. of fused NT- embryos No. (%) of NT-embryos developed to ★ 2-cell 16-cell CM B No. (%) of NT- blastocyst expressed GFP Non- Transfected 67 64 (95.5) 42 (62.7) 39 (58.2) 34 (50.7) 0 Expressed GFP 72 70 (97.2) 41 (56.9) 40 (55.9) 38 (52.8) 37 (97.4) a Unexpressed GFP 74 68 (91.9) 52 (70.3) 43 (58.1) 40 (54.1) 5 (12.5) b a , b Values within columns with different superscripts doffer significantly (p < 0.01). ★ CM: Compacted morula; B: Blastocyst. 86
  67. 68. Fig. Expression GFP of 8-cell stage NT-embryos cloned with adult ear fibroblast cells of which expressing GFP. Bar = 50 μ m. 87 A B C
  68. 69. 88 Fig. Expression pattern of GFP in NT-blastocysts cloned with adult ear fibroblast cells of which expressed GFP. A3: Mosaic GFP expression; B3: Whole GFP expression; A2, B2: Embryos under UV light; A3, B3: Embryos under FITC filters. Bar = 50μm. A1 A2 A3 B3 B2 B1
  69. 70. *Lamb died or was euthanized for animal welfare reasons; PDFF: Poll Dorset fetal fibroblast; M: male; F: female; +: express; -: unexpress; NeO r : neomycin resistant gene; FIX: human blood coagulating factor IX. Table Characteristics of nuclear transfer– derived lambs (Schnieke et al ., 1997) r Induced, unassisted birth F (+) (+) 5.5 155 7LL13 PDFF2-13 11 euthanized at 14 days, heart defect F (+) (+) 3.0 155 7LL16 * Induced, unassisted birth, 7LL16 F (+) (+) 4.6 155 7LL15 PDFF2-12 10 (twins) no breathing Induced, CS24 hours later, heartbeat, F (+) (+) 3.6 148 7LL14 * PDFF2-12 9 stillbirth, one fetus abnormal F (+) (+) 4.5 132 7LL11 * Loss of fetal heartbeat, induced, CS, F (+) (+) 3.6 132 7LL10 * PDFF2-12 8 (twins) Spontaneous abortion F 130 7LL3 * PDFF2-12 7 Induced, CS52 hours later F (-) (+) 8.7 155 7LL12 PDFF2 pool 6 postpartum, meconium in lung Induced, CS52 hours later, died 90 min F (-) (+) 6.3 161 7LL9 * PDFF2 pool 5 lamb Assisted birth because of position of F (-) (+) 7.6 155 7LL8 PDFF2 pool 4 Regressed <80 PDFF2 pool 3 M 3.7 150 7LL7 * Stillbirth, one fetus dead for ≦1week M 3.4 150 7LL6 * PDFF5 2 (twins) Unassisted birth M 3.8 145 7LL5 PDFF5 1 Comments Sex FIX NeO Birth weight (kg) Gestation (days) Lamb Nuclear transfer donor cell type Pregnancy no.
  70. 71. (Chen et al ., 2002) Donor Table Development rates of NT-derived embryos from G418- and GFP-selected cell groups 62 (91.0) b 68 68 (41.2) a 99 (60.0) a 165 (49.8 ) a 331 GFP 36 (47.9) a 73 89 (44.9) a 133 (67.2) a 198 (44.7) a 443 G418 No. transgenic blastocysts (%) No. embryos screened No. blastocysts (%) No. embryos cleaved (%) No. couplets fused (%) No. couplets reconstructed a,b Values with different superscripts within same column differ significantly. (P < 0.001, chi-square test). G418: neomycin, 350 μg/ ml, 14 days. GFP: green fluorescent protein (Clontech; Palo Alto, CA.)
  71. 72. Fig. Analyses of milk production and compositions of somatic clones and matched comparator cows. ( A ) Representative first lactation curves of a clone and a comparator cow. ( B ) Milk total protein (percentage), total fat (percentage), lactose (percentage), total solids (percentage), milk urea nitrogen (mg/dl) and somatic cell count ( 10 3 ml). 複製動物乳肉成分之比較 (Tian et al., 2005)
  72. 73. (Tian et al., 2005)
  73. 74. 體細胞複製技術之應用  具優良遺傳性能種畜之擴增  結合基因轉殖技術建立生物工廠產製人類醫用蛋白  瀕臨絕種動物的復育  成體動物胚幹細胞庫之建立
  74. 75. Challenges of epigenetic reprogramming after transfer of a differentiated donor cell nucleus into an enucleated oocyte.
  75. 76. Thank.. Thank. Thank…

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