B
Animal Reproductive Cloning Data Tables on Reproductive Cloning Efficiency and Defects

The purpose of these tables is to provide an overview of the data from animal cloning experiments done to date (August 2001). Table 1 describes the success/failure rates of reproductive cloning in animals, and Table 2 provides details of the defects or lack of observable defects in reproductively cloned animals. These data were obtained through a comprehensive review of the publications cited in the “Reference” column of each table. Only experiments that yielded live-born cloned offspring were included in the table.

Tables 1 and 2 developed by the panel are supplemented by Tables 3 and 4 developed by Lewis et al., 2001. Note that Tables 3 and 4 use the term “cytoplast” for what the panel calls “enucleated egg.”

How to read Table 1:

Example: The first line from the table can be read as following:

In the experiments described in the paper published by Campbell in 1996 (Column 12), 244 sheep embryos were created using somatic cell nuclear transplantation techniques. The donor nuclei were taken from epithelial-like cells grown from a culture of embryonic stem cells (Column 2). Of these 244 embryos, only 34, or 14%, went on to develop into the morula or blastocyst embryos that are used in the embryo transfer procedure (Column 4). All 34 of those developing embryos were transferred into the wombs of female sheep (as we can tell from Column 8, which indicates



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Scientific and Medical of Aspects: Human Reproductive Cloning B Animal Reproductive Cloning Data Tables on Reproductive Cloning Efficiency and Defects The purpose of these tables is to provide an overview of the data from animal cloning experiments done to date (August 2001). Table 1 describes the success/failure rates of reproductive cloning in animals, and Table 2 provides details of the defects or lack of observable defects in reproductively cloned animals. These data were obtained through a comprehensive review of the publications cited in the “Reference” column of each table. Only experiments that yielded live-born cloned offspring were included in the table. Tables 1 and 2 developed by the panel are supplemented by Tables 3 and 4 developed by Lewis et al., 2001. Note that Tables 3 and 4 use the term “cytoplast” for what the panel calls “enucleated egg.” How to read Table 1: Example: The first line from the table can be read as following: In the experiments described in the paper published by Campbell in 1996 (Column 12), 244 sheep embryos were created using somatic cell nuclear transplantation techniques. The donor nuclei were taken from epithelial-like cells grown from a culture of embryonic stem cells (Column 2). Of these 244 embryos, only 34, or 14%, went on to develop into the morula or blastocyst embryos that are used in the embryo transfer procedure (Column 4). All 34 of those developing embryos were transferred into the wombs of female sheep (as we can tell from Column 8, which indicates

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Scientific and Medical of Aspects: Human Reproductive Cloning number of embryos transferred). Of those 34 embryos, only 8 individual pregnancies resulted (Column 5). Of those 8 pregnancies, 3, or 38%, ended in miscarriage, and 5, or 63%, went on to produce live offspring (Columns 6 and 7, respectively). Of the five lambs that were born alive, only 2 (40%) survived until the time of publication. In all, 2% of the 244 embryos created resulted in live offspring (Column 9), and 12.5% of the 34 embryos transferred into recipient female sheep resulted in live offspring (Column 8). How to read Table 2: Any given line in Table 2 gives an overview of the clinical outcomes of each animal reproductive cloning experiment. For example, in line 1, in the sheep nuclear transplantation experiments published by Campbell in 1996 (Column 7), no information was given concerning the defects seen in miscarried fetuses (Column 3) or about the characteristics of placentas from these pregnancies (Column 6). However, Columns 4 and 5 indicate that 2/5 of the cloned lambs produced in this experiment were healthy and normal, whereas 3/5 died of unknown causes. Note about Figures 1, 2, and 3 Figures 1, 2, and 3 were generated based on data presented in Table 1. Certain experiments whose results are displayed in Table 1 were omitted from the graphs due to incomplete data for all categories displayed in the graphs. Data from reproductive cloning experiments using embryonic, fetal and adult cells as nucleus donors were included in these graphs.

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Scientific and Medical of Aspects: Human Reproductive Cloning Tables and Figures

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Scientific and Medical of Aspects: Human Reproductive Cloning TABLE 1 Rates of Success/Failure of Somatic Cell Nuclear Transfer in Mammals 1 2 3 4 5 6 Speciesa Cell typeb # Embryos producedc # Embryos developed into morula/ blastocyst (%)d # Fetuses after embryo transfere # Fetuses miscarried (%)f Sheep Embryo-derived epithelial-like 244 34 (14) 8 3 (38) Adult mammary gland 277 29 (12) 1 0 (0) Fetal fibroblast 172 47 (27) 5 2 (40) Embryo-derived epithelial-like 385 126 (33) 15 11 (73) Fetal fibroblast 507 69 (13.6) 14 7 (50) ES cell line-derived epithelial-like 128 31 (24.2) >9 >7 (~78) ES cell line-derived epithelial-like 258 44 (17) >11 >10 (~91) ES cell line-derived epithelial-like 423 75 (18) 8 5 (63) ES cell line-derived fibroblast-like 158 39 (31) 10 7 (70) ES cell line-derived fibroblast-like 187 51 (27) 15 8 (53) Fetal fibroblast 417 80 (19) 20 6 (30) Cattle Blastomere (embryonic) 641 152 (24) >13 >4 (~31) Blastomere (embryonic) 132 84 (64) N/A N/A Embryonic stem cell 239 42 (18) N/A N/A Fetal fibroblast 276 33 (12) 6 2 (33) Adult mural granulosa from 13 yr old cow 621 259 (42) 28 26 (93)

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Scientific and Medical of Aspects: Human Reproductive Cloning 7 8 9 10 11 12 # Live births/ Total # fetuses (%)g # Live births/ # Embryos transferred to uterus(%)h # Live births/ # Embryos produced (%)i # Offspring alive or healthy at time of publication/ # Live births (%)j Phenotypes observedk ReferenceL 5/8 (63) 5/34 (12.5) 5/244 (2.0) 2/5 (40) # Campbell 1996 1/1 (100) 1/29 (3.4) 1/277 (.36) 1/1 (100) # Wilmut 1997 3/5 (60) 3/40 (7.5) 3/172 (1.7) 2/3 (67) E# Wilmut 1997 4/15 (27) 4/87 (4.6) 4/385 (1.0) 4/4 (100) # Wilmut 1997 7/14 (50) 7/67 (10.4) 7/507 (1.3) 5/7 (71) BC# Schnieke 1997 2/>9 (<22) 2/31 (6.5) 2/128(1.6) 2/2 (100) CE# Wells 1997 in vivo-matured oocytes 1?>11 (<9) 1/44 (2.3) 1/258 (.39) 0/1 (0) BEF Wells 1997 in vitro-matured ooctyes 3/8 (38) 3/75 (4.0) 3/423 (.7) 2/3 (67) B# Wells 1998n experiment 1 3/10 (30) 3/39 (7.7) 3/158 (1.9) 1/3 (33) B# Wells 1998n experiment 2 7/15 (47) 7/44 (16) 7/187 (3.7) 2/7 (29) BE# Wells 1998n experiment 3 14/20 (70) 14/80 (17.5) 14/417 (3.4) 3/14 (21) E# McCreath 2000 N/A 9/59 (15) 9/641 (1.4) N/A N/A Chesne 1993 N/A 19/78 (24) 19/132 (14) N/A N/A Cheong 1993 N/A 4/34 (12) 4/239 (1.7) N/A N/A Sims 1994 4/6 (67) 4/28 (14.3) 4/276 (1.4) 3/4 (75) ABCF# Cibelli 1998 2/28 (7.1) 2/74 (2.7) 2/621 (.32) 1/2 (50) CD# Wells 1998o

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Scientific and Medical of Aspects: Human Reproductive Cloning 1 2 3 4 5 6 Speciesa Cell typeb # Embryos producedc # Embryos developed into morula/ blastocyst (%)d # Fetuses after embryo transfere # Fetuses miscarried (%)f   Adult cumulus 47 18 (38) 5 0 (0) Adult oviduct epithelial 94 20 (21) 3 0 (0) Adult mural granulosa 552 383 (69) 45 35 (78) Adult mammary gland epithelium 140 36 (26) >2 >1 Adult ear skin fibroblast 82 49 (60) >5 >1 Fetal germ cell 279 85 (30) >17 >16 Fetal fibroblast 174 35 (20) >3 >1 Adult skin cell from ES cell clone 175 N/A 1 0 (0) Adult muscle 346 73 (21) 8 4 (50) Fetal fibroblast 876 >110? (>13) >36 >28 (~78) Adult senescent fibroblast 1896 87 (4.6) >18 >11 (~61), 1 inducedm Adult fibroblast from 17 yr old bull 338 103 (30) 12 6 (50) Many adult and fetal types 1502 596 (40) >50 >26 (~52) Adult and fetal fibroblast N/A N/A >54 >50 (~92) Adult fibroblast from 21 yr old bull 190 53 (28) 6 1 inducedm Mice Adult cumulus 2468 1385 (56) N/A N/A Embryonic stem cell 36 23 (64) N/A N/A Mural trophectoderm 26 16 (62) N/A N/A Adult fibroblast 463 377 (81) N/A N/A Immature adult Sertoli cell 1846 436 (24) 235 219 (93)

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Scientific and Medical of Aspects: Human Reproductive Cloning 7 8 9 10 11 12 # Live births/ Total # fetuses (%)g # Live births/ # Embryos transferred to uterus(%)h # Live births/ # Embryos produced (%)i # Offspring alive or healthy at time of publication/ # Live births (%)j Phenotypes observedk ReferenceL 5/5 (100) 5/6 (83) 5/47 (11) 2/5 (40) # Kato 1998 3/3 (100) 3/4 (75) 3/94 (3) 2/3 (67) # Kato 1998 10/45 (22) 10/100 (10) 10/552 (1.8) 10/10 (100) ABC# Wells 1999 1/>2 (<50) 1/4 (25) 1/140 (.7) 1/1 (1) # Zakhartchenko 1999p 1/>5 (<20) 1/16 (6.3) 1/82 (1.2) 0/1 (0) AG Zakhartchenko 1999p 1/>17 (<6) 1/32 (3.1) 1/279 (.36) 0/1 (0) N/A Zakhartchenko 1999q 2/>3 (<67) 2/7 (29) 2/174 (1.1) 1/2 (50) AB# Zakhartchenko 1999r 1/1 (100) 1/6 (16) 1/175 (.57) 0/1 (0) CD Renard 1999 4/8 (50) 4/26 (15) 4/346 (1.2) 1/4 (25) ABG# Shiga 1999 8/36 (22) 8/110 (7.2) 8/876 (.9) 6/8 (75) BCF# Hill 1999 6/>18 (<33) 6/79 (7.6) 6/1896 (.32) 6/6 (100) ABD# Lanza 2000s 6/12 (50) 6/54 (11) 6/338 (1.2) 4/6 (67) AD# Kubota 2000 24/>50 (<48) 24/172 (14) 24/1502 (1.6) 13/24 (54) ADEG# Kato 2000 4/>54 (<7.4) 4/243 (1.6) 4/? 1/4 (25) BCDF# Hill 2000t 1/6 (17) 1/26 (3.8) 1/190 (.52) 1/1 (100) BD# Hill 2000u N/A 31/1385 (2.2) 31/2468 (1.3) 22/31 (71) # Wakayama 1998 N/A 2/18 (11) 2/36 (5.6) N/A N/A Tsunoda 1998 N/A 2/25 (8) 2/26 (7.7) N/A N/A Tsunoda 1998 N/A 3/274 (1.1) 3/463 (.6) 1/3 (33) BF# Wakayama 1999 16/235 (6.8) 16/436 (3.7) 16/1846 (.87) 15/16 (94) GF# Ogura 2000v

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Scientific and Medical of Aspects: Human Reproductive Cloning 1 2 3 4 5 6 Speciesa Cell typeb # Embryos producedc # Embryos developed into morula/ blastocyst (%)d # Fetuses after embryo transfere # Fetuses miscarried (%)f   Tail tip fibroblast 753 260 (41) 126 119 (94) Adult cumulus 3920 N/A N/A N/A Fetal fibroblast 938 278 (30) 45 40 (89) Adult cumulus (from hybrid strains) 4326 2583 (60) N/A N/A Embryonic gonadal cell 179 114 (64) N/A N/A Embryonic stem cell (from hybrid strain) 783 169 (22) N/A N/A Goat Blastomere (embryonic) 354 96 N/A N/A Fetal fibroblast 230 89 (39) 20 17 (85) Fetal fibroblast 198 157 (79) >6? N/A Pig Adult granulosa >401 401 (?) 9 4 (44) Fetal fibroblast 210 188 (90) N/A N/A Fetal body cell 143 N/A N/A N/A Fetal genital ridge 340 N/A N/A N/A Monkey Blastomere (embryonic) 78 59 (76) 3 1/3 (33) A = High birth weight B = Pulmonary problems C = Cardiovascular abnormalities D = Immune system abnormalities/infection E = Kidney and/or liver abnormalities F = Placental abnormalities G = Joint malformations or other gross deformities # = Healthy offspring produced NOTE: “N/A” indicates that no data were available in the cited publication. NOTE: ES cell = embryonic stem cell. NOTE: (~ ) indicates percentages extrapolated from data available, as shown in other columns. aThe species of animal used in the experiment. bThe cell type used as the source of the donor nucleus for the nuclear transfer.

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Scientific and Medical of Aspects: Human Reproductive Cloning 7 8 9 10 11 12 # Live births/ Total # fetuses (%)g # Live births/ # Embryos transferred to uterus(%)h # Live births/ # Embryos produced (%)i # Offspring alive or healthy at time of publication/ # Live births (%)j Phenotypes observedk ReferenceL 7/126 (5.6) 7/280 (2.5) 7/753 (.93) 7/7 (100) # Ogura 2000w N/A 35/? (>.9%?) 35/3920 (.89) 34/35? (97?) # Wakayama 2000 5/45 (11) 5/272 (1.8) 5/938 (.53) 3/5 (60) BGF# Ono 2001 N/A 80/2573 (3.1) 80/4326 (1.8) N/A # Wakayama 2001 N/A 6/114 (5.2) 6/179 (3.4) 5/6 (83) # Wakayama 2001 N/A 28/? (>16.6?) 28/783 (.36) 22/28 (79) ABF# Eggan 2001 45/? 45/141 (32) 45/354 (13) N/A # Yong 1998 3/20 (15) 3/85 (3.5) 3/230 (1.3) 3/3 (100) # Baguisi 1999 N/A 6/97 (6.1) 6/198 (3.0) 3/6 (50) #D Keefer 2001 5/9 (55) 5/401 (1.2) 5/>401 (<1.2) 5/5 (100) # Polejaeva 2000 N/A 1/110 (.9) 1/210 (.5) 1/1 (100) # Onishi 2000 N/A 2/143 (1.4) 2/143 (1.4) N/A N/A Betthauser 2000 N/A 2/164 (1.2) 2/340 (.59) N/A N/A Betthauser 2000 2/3 (67) 2/29 (6.9) 2/78 (2.6) 2/2 (100) # Meng 1997

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Scientific and Medical of Aspects: Human Reproductive Cloning cThe number of embryos that were successfully formed after the nuclear transfer (cloning) procedure (in the literature usually referred to as # “fused” or # “reconstituted”). In cases where this number was not available, the total number of oocytes injected with nuclei was used (including both successful and failed attempts to produce embryos). In cases of double (serial) nuclear transfer, numbers of successfully reconstructed embryos from the second transfer were used. dThe number and percentage of cloned embryos that continued to develop past the one-cell stage into multicellular embryos called morulae or blastocysts. fThe number of fetuses that were spontaneously aborted at any time during the pregnancy. gThe proportion of pregnancies that were carried to term (comparison of # live births to total # pregnancies). IThe proportion of cloned embryos that went on to become live offspring (comparison of live births to the number of cloned embryos created). jThe survival rate of live born clones after birth (comparison of live born offspring to the number still alive at the time of publication of the reference from which the data were obtained). kThe letters indicate categories of characteristics observed in cloned animals (miscarried, live born or those that died after birth). Categories are provided above, in a key located below the table. LThe peer reviewed scientific article in which data for any given experiment were published. Full references can be found in the bibliography. mFetal miscarriage (abortion) was induced by researchers for medical or research reasons. nWells et al. Cloning sheep from cultured embryonic cells. Reprod. Fertil. Dev. 1998; 10:615-626. oWells et al. Adult somatic cell nuclear transfer is used to preserve the last surviving cow of the Enderby Island cattle breed. Reprod. Fertil. Dev. 1998; 10:369-378. pZakhartchenko et al. Adult cloning in cattle: Potential of nuclei from a permanent cell line and from primary cultures. Mol. Reprod. Fertil. 1999; 54:264-272. qZakhartchenko et al. Potential of fetal germ cells for nuclear transfer in cattle. Mol. Reprod. Dev. 1999; 52:421-426. rZakhartchenko et al. Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts. J. Reprod. Fertil. 1999; 115:325-331. sLanza et al. Extension of cell life-span and telomere length in animals cloned from senescent somatic cells. Science 2000 Apr 28; 288:665-669. tHill et al. Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biol. Reprod. 2000; 63:1787-1794. uHill et al. Development rates of male bovine nuclear transfer embryos derived from adult and fetal cells. Biol. Reprod. 2000; 62:1135-1140. vOgura et al. Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells. Biol. Reprod. 2000; 62:1579-1584. wOgura et al. Birth of mice after nuclear transfer by electrofusion using tail tip cells. Mol. Reprod. Dev. 2000; 57:55-59.

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Scientific and Medical of Aspects: Human Reproductive Cloning FIGURE 1 Survival Rates of Sheep, Cattle and Mouse Embryos Cloned from Adult, Fetal and Embryonic Cells.

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Scientific and Medical of Aspects: Human Reproductive Cloning 5 6 7 Phenotypes of live born clonesf Placental defects, phenotypesg Referencea 15/16 pups normal; 1/16 had umbilical hernia, but was viable at birth unusually large but structurally normal placentas Ogura 2000q 7/7 pups normal, healthy N/A Ogura 2000r telomeres lengthened rather than shortened in successive generations suggesting no inherited aging problem, all mice tested normal for behaviors (learning, memory, activity, agility, strength) N/A Wakayama 2000 5 of the healthy cloned mice were tested for behavioral defects - 3/10 measures of preweaning development were delayed but did appear and had no long-term effects; cloned mice were normal for learning, memory, activity and motor skills - these mice had high postnatal weight gain (not heavy at birth as in LOS) compared to controls but researchers suggest this may have been caused by the agouti gene in their background N/A Tamashiro 2000 3/5 normal and healthy; 2/5 died with umbilical hernia and respiratory deficiency placental hypertrophy and also placental structural abnormalities Ono 2001 28/28 had high birth weights, but this did not adversely affect clones in terms of survival; no respiratory or other problems high placental weights Eggan 2001 45/45 healthy N/A Yong 1998 3/3 normal, healthy N/A Baguisi 1999 3/6 healthy; 3/6 died with respiratory infections placentas within normal range for # of cotyledons Keefer 2001

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Scientific and Medical of Aspects: Human Reproductive Cloning 1 2 3 4 Speciesb Cell typec Defects seen in miscarried fetusesd # Live birthse Pig Adult granulosa N/A 5 Fetal fibroblast N/A 1 Body cell and genital ridge cell N/A 4 Monkey Blastomere (embryonic) N/A 2 NOTE: N/A indicates that no data were available in the cited publication NOTE: ES cell = embryonic stem cell. NOTE: LOS = large offspring syndrome. aThe peer reviewed scientific article in which data for any given experiment were published. Full references can be found in the bibliography. bThe species of animal used in the experiment. cThe cell type used as the source of the donor nucleus for the nuclear transfer. dDescription of abnormalities seen in aborted cloned fetuses; in some cases, these abnormalities may be the cause of miscarriage. eThe number of live-born cloned animals. fDescription of observations of physical, physiological or genetic characteristics of live born cloned animals at time of publication of cited refernces, unless stated otherwise. gDescription of any characteristics, normal or abnormal, noted in the placentas of live born or miscarried cloned animals. hWells et al. Cloning sheep from cultured embryonic cells. Reprod. Fertil. Dev. 1998; 10:615-626. ICibelli et al. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 1998; 280:1256-8. jWells et al. Adult somatic cell nuclear transfer is used to preserve the last surviving cow of the Enderby Island cattle breed. Reprod. Fertil. Dev. 1998; 10:369-378. kZakhartchenko et al. Adult cloning in cattle: Potential of nuclei from a permanent cell line and from primary cultures. Mol. Reprod. Fertil. 1999; 54:264-272. LZakhartchenko et al. Potential of fetal germ cells for nuclear transfer in cattle. Mol. Reprod. Dev. 1999; 52:421-426. mZakhartchenko et al. Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts. J. Reprod. Fertil. 1999; 115:325-331. nLanza et al. Extension of cell life-span and telomere length in animals cloned from senescent somatic cells. Science 2000 Apr 28; 288:665-669. oHill et al. Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biol. Reprod. 2000; 63:1787-1794. pHill et al. Development rates of male bovine nuclear transfer embryos derived from adult and fetal cells. Biol. Reprod. 2000; 62:1135-1140. qOgura et al. Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells. Biol. Reprod. 2000; 62:1579-1584. rOgura et al. Birth of mice after nuclear transfer by electrofusion using tail tip cells. Mol. Reprod. Dev. 2000; 57:55-59.

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Scientific and Medical of Aspects: Human Reproductive Cloning 5 6 7 Phenotypes of live born clonesf Placental defects, phenotypesg Referencea 5/5 pigs very healthy N/A Polejaeva 2000 1/1, Xena, is healthy normal placenta Onishi 2000 no phenotypes described N/A Betthauser 2000 2/2 healthy N/A Meng 1997

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Scientific and Medical of Aspects: Human Reproductive Cloning TABLE 3 Developmental Capacity of Cytoplasts Reconstituted with Nuclei from Embryonic Cells 1 2 3 4 5 Species Recipient cytoplast Donor cell type % Early Development: % Blastocyst (# Blastocysts/ # Cultured) % Term Development: % Offspring (# Live births/ # Transferred) Mouse Zygote Inner cell mass 16% (23/142) 19% (3/16) Trophectoderm 1% (1/68) 0 Zygote Pronuclear 95% (20/21) no transfer 2-cell 13% (19/151) no transfer 4-cell 0 (0/81) no transfer 8-cell 0 (0/111) no transfer Inner cell mass 0 (0/84) no transfer Zygote 8-cell 0(0/32) no transfer Inner cell mass 0 (0/84) no transfer Zygote 8-cell 0(0/32) no transfer 2-cell blastomere 2-cell 93% (40/43) 24%a (10/41) Zygote 8-cell 51% (45/89) 0a (0/11) Cumulus cell 0 (0/91) no transfer 2-cell blastomere 4-cell 72% (49/68) 22% (10/46) 8-cell 35% (49/139) 8% (4/48) Inner cell mass 0 (0/91) no transfer Mll oocyte 2-cell 23% (20/88) 15% (3/20) 8-cell 4% (1/26) 0 (0/1) Inner cell mass 13% (11/87) 0 (0/11) 2-cell 78% (36/46) 29% (10/34) 4-cell 71% (30/42) 22% (6/27) 8-cell 46% (18/39) 18% (3/17) 4-cellc 83% (58/70) 43% (2/58) Inner cell massc 64% (23/36) 11% (2/18) Trophectodermc 62% (16/26) 8% (2/25) ES cell 5% (47/931) 0 (0/56) ES cell 29% (312/1087) 6% (8/132) Sheep Mll oocyte 8-cell 33% (8/24) 75% (3/4)   16-cell 27% (13/49) 21% (3/14)

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Scientific and Medical of Aspects: Human Reproductive Cloning 6 7 References Significant findings Illmensee 1981 First demonstration of developmental potential in mammals. Reproducibility of results questioned. McGrath 1984 Biologically impossible to achieve development with transcriptionally active nucleus. Robl 1986 Development more advanced with cytoplast prepared from 2-cell than zygote. Robl 1986 Development more advanced with cytoplast prepared from 2-cell than zygote. No development beyond 12 days gestation. Wakayama 2000b No development when zygotic cytoplasts were used. Robl 1987 Term development when 4- and 8-cell nuclei used but not more advanced. Importance of cytoplast environment. Kono 1991g Development to term from embryonic nuclei transferred to enucleated oocyte. Cheong 1993 Embryonic nuclei in G1 phase of the cell cycle can direct term development when transferred to Mll cytoplasts. Kwon 1996 Serial nuclear transfer of metaphase-arrested embryonic nuclei results in term development. Tsunoda 1998 Term development following serial nuclear transfer of inner cell mass and trophectoderm nuclei. Tsunoda 1993 Implantation sites but no term development. Wakayama 1999f Late-passage actively dividing ES cell nuclei are able to direct development to term. Willadsen 1986 Term development from cleavage stage blastocysts. Smith 1989 Transcriptionally active nuclei are able to direct development to term.

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Scientific and Medical of Aspects: Human Reproductive Cloning 1 2 3 4 5 Species Recipient cytoplast Donor cell type % Early Development: % Blastocyst (# Blastocysts/ # Cultured) % Term Development: % Offspring (# Live births/ # Transferred)   Inner cell mass 38% (6/16) 11% (1/9)d Cultured cell line 14% (34/244) 14% (5/34) Cattle Pronuclear Pronuclear 13% (5/38) 100% (2/2) 2- to 8-cell 0 (0/10) no transfer Mll oocyte 2- to 8-cell 12% (13/111) 0 (0/12) 9- to 16-cell 16% (8/50) 28 (2/7) 17- to 32-cell 8% (2/24) no transfer Morula (64-cell) 23-35% 22%e (104/463) Morula (31-cell) 24% (152/641) 15% (9/59) Inner cell mass 7% (20/304) 13% (2/15) Inner cell mass 5% (30/629) 8% (2/26) Cultured inner cell mass 27% (109/406) 12% (4/34) Fetal germ cell (PGC) 20-38% (30/149-53/140) 5% (1/20) Rabbit Mll oocyte 8-cell not assessed 4% (6/164) 8- to 16-cell 49% (34/69) 21% (23/110) 32-cell 33% (14/43) 15% (10/67) Inner cell mass 20% (17/83) no transfer Trophectoderm 0 (0/52) no transfer Pig Mll oocyte 2-cell 9% (1/11) 0 (0/33) 4-cell 8% (7/83) 3% (1/34) 8-cell 19% (11/57) 0 (0/21) Goat Mll oocyte Morulac 31% (18/57) 31% (45/141) Monkey Mll oocyte 8-cell 52%e (53/101) 4% (2/53) aDevelopment assessed at 8.5 days post coitum. bWakayama et al. Nuclear transfer into mouse zygotes. Nat Genet 2000; 24:108-9. cAchieved using serial nuclear transfer. dDevelopment assessed at 42 days of pregnancy. eEmbryonic development assessed at the 2-cell stage prior to transfer. fWakayama et al. Mice cloned from embryonic stem cells. Proceedings of the National Academy of Sciences, USA 1999; 96:14984-89.

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Scientific and Medical of Aspects: Human Reproductive Cloning 6 7 References Significant findings Campbell 1996 Nuclei from cell lines from embryonic discs are able to support development to term. Robl 1987 Cleavage stage embryonic nuclei are unable to direct embryonic or term development when transferred to enucleated zygotes. Prather 1987 Term development from transcriptionally active donor embryonic nuclei. Bondioli 1990 Nuclei from morula stage embryos can direct midgestation development. Chesne 1993 Nuclei from morula stage embryos can direct development to term. Collas 1994 Direct injection of inner cell mass nuclei into Mll cytoplasts can direct development to term. Keefer 1994 Totipotency of inner cell mass nuclei confirmed. Sims 1984 Nuclei from inner cell mass cultured for up to 28 days are able to direct development to term. Zakhartchenko 1999 Fetal germ cells can direct development to term. Stice 1988 First production of genetically verified nuclear transfer rabbits from embryonic donor nuclei. Prather 1989 High rates of development from transcriptionally active embryonic nuclei. Collas 1990; Callas 1991 Normal embryonic development from inner cell mass donor nuclei. Prather 1989 Cleavage stage embryonic nuclei can direct term development in pigs. Yong 1998 Serial nuclear transfer of transcriptionally nuclei results in high rates of development. Meng 1997 Embryonic nuclei can support term development in the monkey. gKono T et al. Development of enucleated mouse oocytes reconstituted with embryonic nuclei. J. Reprod. Fertil. 1991; 93:165-72. SOURCE: Lewis, IM, MJ Munsie, AJ French, R Daniels and AO Trounson, 2001. The Cloning Cycle: From Amphibia to Mammals and Back. Reproductive Medicine Reviews 9:1 pp. 3-33.

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Scientific and Medical of Aspects: Human Reproductive Cloning TABLE 4 Developmental Capacity of Cytoplasts Reconstituted by Nuclei from Fetal and Adult Somatic Cells 1 2 3 4 Species Recipient cytoplast Donor Cell Type % Early Development: % Blastocyst (# Blastocysts/ # Cultured) Mouse Zygote Mll oocyte Cumulus Cell Thymocyte 0 (0/91) 7% (6/88) Cumulus Cell 67% (101/151) Neuronal Cell 22% (50/223) Sertoli cell (mature) 40% (63/159) Cumulus Cell 20% (19/93) Cultured follicular cell; 34% (51/151) 3% (1/30) Adult Male fibroblast; 50% (207/414) Cumulus cell. 52% (206/393) Fibroblast cell 23% (38/162) Sertoli Cell (immature) 33% (94/284) Sheep Mll oocyte Fetal fibroblast 27% (34/124) Adult Mammary (epithelial) 12% (29/247) Transgenic Fetal fibroblast 5-21% (5/82-19/89) Transgenic Fetal fibroblast 6-28% (14/109, 43/154, 4/71, 19/83) Cattle Mll oocyte Cumulus cell 13% (5/38) Fetal fibroblast 12% (33/276)

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Scientific and Medical of Aspects: Human Reproductive Cloning 5 6 7 % Term Development: % Offspring / (# Live births/ # Transferred) References Significant Findings No Transfer 0 1. Wakayama 2000a -same footnote information as earlier Wakayama 2000 in Table 3 2. Callas, 1992 1. No development when zygotic cytoplasts were used. 2. Somatic nuclei are able to direct embryonic development through no term development. 2% (31/1315) Wakayama 1998b Direct-injected cumulus cell nuclei direct term development; however, Sertoli and neuronal nuclei do not. 2%a (1/46)   Findings do not support the requirement of G0/G1 nuclei for term development. 2%a (1/59) 0 (0/3) Kato 1999 Serial nuclear transfer of cultured follicular cells but not cumulus cell nuclei results in term development. nuclei can direct term development. 3% (1/30); Wakayama 1999c; Male-derived adult somatic cell nuclei can direct term development; Immature, actively dividing Sertoli cell 1% (2/177); Ogura 2000d 1%c (2/206)   0c (0/38)     4% (6/134)     8% (3/40) Wilmut 1997 Inducing cell to enter quiescence by serum starvation may assist in nuclear reprogramming. 3% (1/29)   First demonstration that nuclei from differentiated somatic fetal or adult origin can direct development to term. 5/21% (1/21-1/5) Schnieke 1997 Term development of transfected somatic cell nuclei suggests an alternative method for the production of transgenic animals. One male lamb was born. 0-28% (4/14, 8/43, 0/4, 2/19) McCreath 2000 Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. 0 (0-19) Callas 1994 Nuclei from adult somatic cells can direct embryonic development. 14% (3/28) Cibelli 1998e Cultured activity dividing fetal fibroblast nuclei can direct development to term.

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Scientific and Medical of Aspects: Human Reproductive Cloning 1 2 3 4 Species Recipient cytoplast Donor Cell Type % Early Development: % Blastocyst (# Blastocysts/ # Cultured)   Adult Male Fibroblast 21-37% (24/114 - 43/115) Cumulus Cell 49% (18/37) Cultured Oviductal cell 23-34% (196/842 - 29/84) Cultured Granulosa 28% (152/552) Rabbit Mll oocyte Adult granulosa Number not specified Pig Mll oocyte Granulosa cell line Not assessed     Fetal fibroblast 1-31% (total 88/615) 93% (2-, 4- and 8-cell, 110/118) Goat Mll oocyte Transgenic Fetal fibroblast Not assessed Monkey Mll oocyte Fetal fibroblast 57% (57/100)     Adult fibroblast 44% (4/9) SOURCE: Lewis, IM, MJ Munsie, AJ French, R Daniels and AO Trounson, 2001. The Cloning Cycle: From Amphibia to Mammals and Back. Reproductive Medicine Reviews 9:1 pp. 3-33. NOTE: Cytoplast = Enucleated Egg. aWakayama et al. Nuclear Transfer into mouse zygotes. Nat Genet 2000; 24:108-9. bWakayama et al. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 1998; 394:369-74. cWakayama T. and Yanagimachi R. Cloning of male mice from adult tail-tip cells. Nat Genet 1999; 22:127-8 dOgura A, et al. Production of male cloned mice from fresh, cultured and cryopreserved immature Sertoli cells. Biol. Reprod. 2000; 62:1579-84. eCibelli J., et al. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 1998; 280:1256-8.

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Scientific and Medical of Aspects: Human Reproductive Cloning 5 6 7 % Term Development: % Offspring / (# Live births/ # Transferred) References Significant Findings 7% (2/7) Kubota 2000 Nuclei from male adult fibroblast can direct development to term. 33% (2/6) Kato 1995 High rates of term development following transfer of cumulus and oviduct nuclei. 50% (2/4)     10% (10/100) Wells 1999 Production of calves from cultured granulosa cells. 0 Collas and Rob, unpublished First production of genetically verified nuclear transfer rabbits. 1.3% (5/401) Polejaeva 2000 Term development following serial nuclei transfer of cumulus cells. No Transfer 0.9% (1/110) Onishi 2000 Term development following direct injection of nuclei from fetal fibroblast cells. 3% (3/112) Baguisi 1999 Production of transgenic goats from transfected fetal fibroblast nuclei. 0 Wolf 1999 Donor nuclei from cell lines are capable of limited embryonic development. 0