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. 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
17. 電 穿孔 / 融合 之設備 (Equipments for Electroporation/ fusion)
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
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
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. 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. 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. 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)
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. Table . Effects of activation agents or chemicals in oocyte activation ( 劉 , 2000)
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. ( 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. ( 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. The interaction of cell cycle between recipient oocyte and donor cell 48
37. M G2 S G1 G0 Growth factor absent Growth factor present Cell cycle stage 2N 2~4N 4N 4N 49
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. 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. 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. 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. 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. (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. (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).
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
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
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
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
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) 。
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
66. Fig. Donor cells with GFP expression. A: Cells under bright light; B: Cells under FITC filters. Bar = 50 μ m. 76 A B
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
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
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
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.
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.)
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)