Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

Genetically modified animals have many poten-tial applications in basic research, human medicine and agriculture. Pronuclear DNA microinjection has been almost the only practical means of producing transgenic animals during the last 20 years, but the low efficiency (1%—5%)[1] of this method has actu-ally been the obstacle that hampered its further appli-cation in animal biotechnology. The birth of Dolly[2], the first somatically cloned animal, made it possible to produce transgenic animals b…     

Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 μg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, respectively). The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal oviduct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo developmental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.     

奶山羊转基因供核细胞的再饥饿对核移植胚胎发育的影响

为提高转基因奶山羊体细胞核移植胚胎早期发育率,将经转染外源基因的山羊胎儿成纤维细胞经饥饿培养(含0．5％FCS的DMEM)5天后分成两部分：第一部分细胞-80℃或液氮冻存,试验前复苏后直接用作供核细胞(试验组Ⅰ),或复苏后恢复培养(含10％FCS的DMEM)2-5天后再饥饿5天用作供核细胞(试验组Ⅱ);第二部分细胞作传代培养(含10％FCS的DMEM)2天后再饥饿5天用作供核细胞(试验组Ⅲ)。将上述不同处理的供核细胞进行细胞周期与存活率的检测,并将该供核细胞移入去除遗传物质的山羊MⅡ期卵母细胞的卵周隙内,经电融合、化学激活后,将核移植(NT)胚胎经0．8％琼脂糖包理后移入临时寄母输卵管内,培养6天后回收并观察NT胚胎的早期发育。结果,试验组Ⅱ所用供核细胞中G0／G1期细胞所占比例及其存活率分别为95．68％、99．9％,均显著地高于试验组Ⅰ(88、66％、80％);试验组Ⅱ的桑椹及囊胚期NT胚胎的发育率(66．09％)显著地高于试验组Ⅰ(22．00％)与试验组Ⅲ(50．5l％)。将以上发育的NT胚胎分别移入同步发情的受体后,35天作B超妊娠诊断,试验组Ⅱ的受体妊娠率为45．83％,显著地高于试验组Ⅰ(20．00％)与试验组Ⅲ(29．58％)。流式细胞仪分析结果表明,饥饿后的供核细胞经冷冻,复苏后恢复培养2-5天,再经饥饿处理,能显著地提高G0／G1期细胞的比例及细胞存活率;应用该细胞所组建的NT胚不仅具有较高的桑椹与囊胚期发育率,而且具有较高的受体妊娠率。     

Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts

The efficiency of nuclear transfer (NT) using two primary cultures of fetal fibroblasts (FF1 and FF2) was compared vs. the same cultures transfected with an expression vector in which the bovine prochymosin coding sequence is placed under the control of the bovine alpha(S1)-casein promoter (TFF1 and TFF2). In addition, fibroblasts of a cloned transgenic fetus (TRFF1) derived from TFF1 and ear skin fibroblasts of a 1-month-old cloned transgenic calf (TRCF1) derived from TRFF1 were used as nuclear donors. Embryos reconstructed from FF1 (44%) and FF2 (52%) developed to the blastocyst stage at a significantly (P < 0.05) higher rate than those derived from TFF1 (24%) and TFF2 (27%). The proportions of cleaved embryos and blastocysts were significantly (P < 0.05) higher with TRFF1 than with TRCF1 used as nuclear donors (75 vs. 66% and 33 vs. 16%, respectively). Transfer of NT embryos derived from FF2 and TFF2 to recipients resulted in similar pregnancy rates on day 30 (52 and 48%, respectively). However, with TFF2 embryos, the majority of pregnancies (8/11; 73%) was lost in the first and second trimesters of gestation, whereas 4/11 (36%) pregnancies with FF2 embryos were lost during the full period of in vivo development. Of 11 FF2 and 6 TFF2 born calves (25 and 13% of transferred embryos, respectively), 6 and 3 survived including one oversized FF2 calf. After transfer of TRFF1 and TRCF1 NT embryos to recipients, initial pregnancy rate was as a tendency higher in the TRFF1 (49%) than in the TRCF1 group (30%). The majority (14/17) of TRFF1 pregnancies and all TRCF1 pregnancies were lost in the first and second trimester. A high proportion of TRFF1 calves (5/8) showed increased body weights, and only two calves which were also large survived. These findings demonstrate that (i) extended culture associated with transfection and selection procedures may induce changes of donor cells which markedly decrease the efficiency of nuclear transfer and (ii) these changes are not reversed by recloning.     

Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.     

Development rates of male bovine nuclear transfer embryos derived from adult and fetal cells

This study compared the nuclear transfer (NT) embryo development rates of adult and fetal cells within the same genotype. The adult fibroblast cells were obtained from a 21-yr-old Brahman bull. The fetal cells were derived from a Day 40 NT fetus previously cloned using cells from the Brahman bull. Overall, similar numbers of blastocysts developed from both adult (53 of 190; 28%) and fetal (39 of 140; 28%) donor cells. Improved blastocyst development rates were observed when fetal cells were serum-starved (serum-fed 12% vs. serum-starved 43%; P < 0.01) whereas there was no similar benefit when adult cells were serum-starved (both serum-fed and serum-starved 28%). Day 30 pregnancy rates were similar for blastocysts derived from adult (6 of 26; 23%) or fetal (5 of 32; 16%) cells. Day 90 pregnancy rates were 3 of 26 for adult and 0 of 32 for the fetal cell lines. One viable bull calf derived from a 21-yr-old serum-starved adult skin fibroblast was born in August 1999. In summary, somatic NT embryo development rates were similar whether adult or fetal cells, from the same genotype, were used as donor cells. Serum starvation of these adult donor cells did not improve development rates of NT embryos to blastocyst, but when fetal cells were serum-starved, there was a significant increase in development to blastocyst.     

Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors and in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes and oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (47.5% vs. 71.6%, p<0.05). For the blastocyst rate, no significant difference was observed between two groups (10% vs. 10.4%, p>0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36 to 42 h (83.6% vs. 96.7%, p<0.05). However, no statistical difference was observed between NTEs derived from oocytes of 36 h IVM group and NTEs from oocytes of 42 h IVM group in the rates of cleavage (59.3% vs. 73.6%, p>0.05) and blastocyst formation (9.3% vs. 13.2%, p>0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O₂) tension and NTEs derived from oocytes matured under higher oxygen tension (20% O₂) in cleavage rate (70.6% vs. 67.1%, p>0.05) and blastocyst rate (11.8% vs. 12.3%, p>0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of MII oocytes as recipients. (iii) Lower oxygen tension and higher oxygen tension in IVM have no significant effect on development of cloned embryos.     

Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors and in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes and oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (45.7% vs. 71.6%, p<0.05). For the blastocyst rate, no significant difference was observed between two groups (10% vs. 10.4%, p>0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36h to 42h (83.6% vs. 96.7%, p<0.05). However, no statistical difference were observed between NTEs derived from oocytes of 36 h IVM group and NTEs from oocytes of 42 h IVM group in the rates of cleavage (59.3% vs. 73.6%, p>0.05) and blastocyst formation (9.3% vs. 13.2%, p>0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O₂) tension and NTEs derived from oocytes matured under higher oxygen tension (20% O₂) in cleavage rate (70.6% vs. 67.1%, p>0.05) and blastocyst rate (11.8% vs. 12.8%, p>0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of MII oocytes as recipients. (iii) Lower oxygen tension and higher oxygen tension in IVM have no significant effect on development of cloned embryos.     

Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT), combined with genome modification techniques, is a very pow-erful tool for agriculture, medicine and fundamental research on basic biological mechanisms. The effi-ciency of producing transgenic animals is greatly prom…     

Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors and in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes and oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (45.7% vs. 71.6%, p<0.05). For the blastocyst rate, no significant difference was observed between two groups (10% vs. 10.4%, p>0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36h to 42h (83.6% vs. 96.7%, p<0.05). However, no statistical difference were observed between NTEs derived from oocytes of 36 h IVM group and NTEs from oocytes of 42 h IVM group in the rates of cleavage (59.3% vs. 73.6%, p>0.05) and blastocyst formation (9.3% vs. 13.2%, p>0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O₂) tension and NTEs derived from oocytes matured under higher oxygen tension (20% O₂) in cleavage rate (70.6% vs. 67.1%, p>0.05) and blastocyst rate (11.8% vs. 12.8%, p>0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of MII oocytes as recipients. (iii) Lower oxygen tension and higher oxygen tension in IVM have no significant effect on development of cloned embryos.     

Generation of human lactoferrin transgenic cloned goats using donor cells with dual markers and a modified selection procedure

The objective was to use dual markers to accurately select genetically modified donor cells and ensure that the resulting somatic cell nuclear transfer kids born were transgenic. Fetal fibroblast cells were transfected with dual marking gene vector (pCNLF-ng) that contained the red-shifted variant of the jellyfish green fluorescent protein (LGFP) and neomycin resistance (Neo) markers. Cell clones that were G418-resistant and polymerase chain reaction-positive were subcultured for several passages; individual cells of the clones were examined with fluorescence microscopy to confirm transgenic integration. Clones in which every cell had bright green fluorescence were used as donor cells for nuclear transfer. In total, 86.7% (26/30) cell clones were confirmed to have transgenic integration of the markers by polymerase chain reaction, 76.7% (23/30) exhibited fluorescence, but only 40% (12/30) of these fluorescent cell clones had fluorescence in all cell populations. Moreover, through several cell passages, only 20% (6/30) of the cell clones exhibited stable LGFP expression. Seven transgenic cloned offspring were produced from these cells by nuclear transfer. Overall, the reconstructed embryo fusion rate was 76.6%, pregnancy rates at 35 and 60 days were 39.1% and 21.7%, respectively, and the offspring birth rate was 1.4%. There were no significant differences between nuclear transfer with dual versus a single (Neo) marker (overall, 73.8% embryo fusion rate, 53.8% and 26.9% pregnancy rates, and 1.9% birth rate with five offspring). In conclusion, the use of LGFP/Neo dual markers and an optimized selection procedure reliably screened genetically modified donor cells, excluded pseudotransgenic cells, and led to production of human lactoferrin transgenic goats. Furthermore, the LGFP/Neo markers had no adverse effects on the efficiency of somatic cell nuclear transfer.     

Developmental potential of bovine nuclear transfer embryos and postnatal survival rate of cloned calves produced by two different timings of fusion and activation

We compared developmental potential of somatic cell nuclear transfer (NT) embryos and postnatal survivability of cloned calves produced by two different fusion and activation protocols. As donor cells for NT, bovine cumulus cell-derived cultured cells of passage 5 were used following culture in serum-starved medium for 5-7 days. Enucleated oocytes were fused with donor cells at 21 or 24 hr post maturation. NT embryos fused at 21 hr were activated chemically 3 hr after fusion (DA group) and embryos fused at 24 hr were activated chemically immediately after fusion (FA group). Chemical activation was accomplished by calcium ionophore for 5 min and cytochalasin D + cycloheximide for 1 hr then cycloheximide alone for 4 hr. After in vitro culture in IVD101 medium for 7 days, embryo transfer was performed. Fusion rates were 86 and 84% in the DA and FA groups, respectively. Developmental rate to the blastocyst stage of NT embryos in the DA group was higher than in the FA group (42% vs. 28%). Pregnancy rate did not differ significantly between the DA and FA groups (11/13 and 5/7 at day 35), and 13 cloned calves (including 1 set of twins from a single embryo transfer) were born. High rates of postnatal mortality were observed in both groups. These results suggest that the DA method improves in vitro developmental potential of NT embryos, but the timing of fusion and chemical activation does not affect the pregnancy rate and the survivability of cloned calves.     

Transgenesis and nuclear transfer using porcine embryonic germ cells

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). Porcine EG cell lines with capacities of both in vitro and in vivo differentiation have been established. Because EG cells can be cultured indefinitely in an undifferentiated state, they may be more suitable for nuclear donor cells in nuclear transfer (NT) than somatic cells that have limited lifespan in primary culture. Use of EG cells could be particularly advantageous to provide an inexhaustible source of transgenic cells for NT. In this study the efficiencies of transgenesis and NT using porcine fetal fibroblasts and EG cells were compared. The rate of development to the blastocyst stage was significantly higher in EG cell NT than somatic cell NT (94 of 518, 18.2% vs. 72 of 501, 14.4%). To investigate if EG cells can be used for transgenesis in pigs, green fluorescent protein (GFP) gene was introduced into porcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts (29 of 137, 21.2%) expressing GFP based on observation under fluorescence microscope. The results obtained from the present study suggest that EG cell NT may have advantages over somatic cell NT, and transgenic pigs may be produced using EG cells.     

Fate of donor mitochondrial DNA in cloned bovine embryos produced by microinjection of cumulus cells

This study examined the fate of donor mitochondrial DNA during preimplantation development after nuclear transfer (NT) in cattle. Frozen-thawed cumulus cells were used as donor cells in the nuclear transfer. Mitochondrial DNA heteroplasmy in the nuclear transfer embryos was analyzed by allele-specific PCR (AS-PCR), direct DNA sequencing, and DNA chromatography. AS-PCR analysis for the detection of donor mitochondrial DNA was performed at the 1-, 2-, 4-, 8-, 16-cell, morula, and blastocyst stages of the embryos. The mitochondrial DNA from donor cells was detected at all developmental stages of the nuclear transfer embryos. However, mitochondrial DNA heteroplasmy was not observed in direct DNA sequencing of displacement-loop sequence from nuclear-transfer-derived blastocyst embryos. To confirm the mtDNA heteroplasmy in cloned embryos, the AS-PCR product from NT-derived blastocysts was analyzed by DNA sequencing and DNA chromatography. The nucleotides of NT-derived blastocysts were in accordance with the nucleotides from donor cells. These results indicate that the foreign cytoplasmic genome from donor cells was not destroyed by cytoplasmic events during preimplantation development that followed nuclear transfer.     

单、双标记基因筛选的转人乳铁蛋白基因供核细胞生产克隆山羊效率的比较

为比较两种筛选标记基因生产转人乳铁蛋白(hLF)基因克隆山羊的效率,利用单(新霉素抗性基因,Neor)、双(新霉素抗性和绿色荧光蛋白基因,Neor/GFP)标记基因筛选转基因的供核细胞,并制作体细胞核移植转基因山羊。山羊胎儿成纤维细胞电转染单标记基因表达载体(pBLC14)或双标记基因表达载体(pAPLM),分别有58.8%(20/34)和86.7%(26/30)的抗性细胞株检测到外源基因;转染pAPLM的细胞传代培养后,仅有20%(6/30)株细胞在传代中所有细胞均能观察到荧光;分别以pBLC14和pAPLM的细胞株作为供核细胞进行体细胞核移植,共获得806枚重构胚胎,胚胎移植受体后35 d、60 d妊娠率分别为53.8%、26.9%和39.1%、21.7%,最终分别产下5只(1.9%)和7只(1.4%)克隆山羊;经PCR及Southern blotting检测,所有出生山羊均整合有外源基因。结果显示,以单、双标记基因筛选供核细胞,其重构胚融合率、怀孕率和克隆动物出生率差异不显著(P>0.05),Neor/GFP双标记基因能准确、有效地用于转基因供核细胞筛选。同时,结果也表明Neor/GFP双标记基因转染的体细胞作为供核细胞对体细胞克隆效率未出现不利影响。     

Characterization of a donor mitochondrial DNA transmission bottleneck in nuclear transfer derived cow lineages

In embryos derived by nuclear-transfer (NT), fusion of donor cells with recipient oocytes resulted in varying patterns of mitochondrial DNA (mtDNA) transmission in NT animals. Distribution of donor cell mtDNA (D-mtDNA) found in offspring of NT-derived founders may also vary from donor cell and host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to G(1) offspring. Eleven NT founder cows were produced by fusion of enucleated oocytes (Holstein/Japanese Black) with Jersey/ Holstein oviduct epithelial cells, or Holstein/Japanese Black cumulus cells. Transmission of mtDNA was analyzed by PCR mediated single-strand conformation polymorphism of the D-loop region. In six of seven animals sampled postmortem, heteroplasmy were detected in various tissues, while D-mtDNA could not be detected in blood or hair samples from four live animals. The average proportion of D-mtDNA detected in one NT cow was 7.6%, and those in other cows were <5%. Heteroplasmic NT cows (n = 6) generated a total 12 G(1) offspring. Four of 12 G(1) offspring exhibited high percentages of D-mtDNA populations (range 17-51%). The remaining eight G(1) offspring had slightly or undetectable D-mtDNA (<5%). Generally, a genetic bottleneck in the female germ-line should favor a homoplasmic state. However, proportions of some G(1) offspring maintained heteroplasmy with a much higher percentage of D-mtDNA than their NT dams, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrate that D-mtDNA in NT cows is transmitted to G(1) offspring with varying efficiencies.     

Production of cloned goats by nuclear transfer of cumulus cells and long-term cultured fetal fibroblast cells into abattoir-derived oocytes

Dairy goats are ideal for the transgenic production of therapeutic recombinant proteins. The use of recombinant somatic cell lines for nuclear transfer (NT) allows the introduction of genes by transfection, increases the efficiency of transgenic animal production to 100%, and overcomes the problem of founder mosaicism. Although viable animals have been cloned via NT from somatic cells of 11 species, the efficiency has been extremely low. Both blastomere and somatic cell NT increased fetal loss and perinatal morbidity/mortality in cattle and sheep, but fetal loss and perinatal mortality appear to be relatively low in goats. In this study, we produced cloned goats by NT from cumulus cells and long-term cultured fetal fibroblast cells (FFCs) to abattoir-derived oocytes. NT embryos were constructed from electrofusion of cumulus cells (CCs), FFCs, or skin fibroblast cells (SFCs) with cytoplasts prepared from abattoir-derived ovaries. The NT embryos were activated with an optimized activating protocol (1 min exposure to 2.5 microM ionomycin followed by 2 hr incubation in 2mM 6-DMAP). Two viable cloned kids from CCs and one from long-term cultured FFCs (at passage 20-25) were born. Microsatellite analysis of 10 markers confirmed that all cloned offspring were derived from corresponding donor cells. To our knowledge, the production of cloned goat offspring using abattoir-derived oocytes receiving nuclei from CCs and long-term cultured FFCs has not been reported. The production of viable cloned animals after activation with reduced intensity of ionomycin and 6-DMAP treatment has also not been reported. Loss of cloned embryos was obvious after 45 and 90 days of pregnancy, and a lack of cotyledons, heart defects, and improperly closed abdominal wall were observed in the aborted fetuses and one cloned kid. The fusibility and in vitro developmental potential of embryos reconstructed from FFCs at passage 20-25 were significantly lower than those of embryos reconstructed from FFCs at passage 3-5, and the cloning efficiency of the long-term cultured cells was low (0.5%).