整合人lactoferrin基因的山羊体细胞支持核移植克隆胚的体外发育

克隆了人lactoferrin基因和山羊[[beta]]-casein基因5′端调控区, 构建了人lactoferrin的乳腺表达载体, 并将该载体利用脂质体介导转染了奶山羊胎儿成纤维细胞, 获得了稳定整合人lactoferrin基因的转基因体细胞克隆17个, 其中PCR和Southern Blot检测阳性的细胞克隆14个, 阳性率82.4%。以转基因体细胞为供体细胞进行了核移植, 获得了能够体外发育的山羊转基因克隆胚胎, 体内成熟卵母细胞来源的核移植囊胚率为64.8%, 体外成熟卵母细胞来源的核移植囊胚率为51.7%, 证明了山羊转基因体细胞能够支持克隆胚的进一步发育。     

重组人SOD1/3转基因山羊的制备及表达产物的检测

设计了以hSOD1、hSOD3为编码序列,以山羊β-酪蛋白/CMV杂合启动增强子构建乳腺特异性表达载体rhSOD1、rhSOD3,共转染母山羊胎儿成纤维细胞,采用PCR和扩增产物序列分析筛选获得SOD1/3克隆细胞株,应用体细胞核移植(SCNT)制备双转基因山羊。出生小羊经PCR和扩增产物序列分析验证是否成功整合外源基因,经Western blotting、ELISA及体外活性检来验证分析表达产物。结果表明:获得SOD1/3转基因山羊胎儿成纤维细胞系6株;原代双转基因体克隆山羊1只(♀);从该转基因羊乳汁中检测到rhSOD1、rhSOD3,浓度分别为:88.81±8.36 mg/L和267.82±12.67 mg/L;转基因羊乳汁中重组人SOD酶活性为1 432±157 U/mL。研究表明,以双载体和单标记基因转染山羊胎儿成纤维细胞可获得双基因整合转基因细胞系,并且以SOD1与SOD3功能基因均可在山羊乳腺中共同表达,表达产物具有较好的生物学活性。     

ht-Pam基因在山羊β-酪蛋白基因座定位整合的研究

利用体细胞基因打靶与核移植技术制备动物乳腺生物反应器是当今转基因定位整合表达的一种新技术。分别克隆山羊的β-酪蛋白基因5′调控区的6.3kb片段,外显子7、外显子8和9三个基因片段,并与克隆的人tPA突变体cDNA一起构建了含有neo和tk正负筛选标记基因的β-酪蛋白基因打靶载体P^GBC4tPA,并验证了neo基因、tk基因以及Cre-LoxP系统的有效性。将线性化的P^GBC4tPA通过电转染整合到山羊胎儿成纤维细胞基因组中,利用G418和GANC进行抗性细胞克隆的药物筛选,初步获得抗性细胞克隆244个,PCR检测后获得阳性细胞克隆31个,其中初步验证2个细胞克隆转植基因整合位点重组后的基因序列正确,并且该细胞克隆能够有效扩增。这为下一步基因打靶体细胞核移植制备山羊乳腺生物反应器奠定了基础。     

以经过转染的乳腺上皮细胞生产克隆羊

为研究转基因乳腺上皮细胞发育的全能性,利用电转染方法将人乳铁蛋白(hLF)乳腺特异性表达载体电转染山羊乳腺上皮细胞,经G418和PCR筛选获得阳性克隆细胞株,经催乳素诱导的细胞株上清液用Western blotting方法检测hLF的表达。以转基因与上清液中表达hLF均为阳性的细胞为核供体细胞,进行山羊体细胞核移植。结果为:16株细胞表达重组hLF,分子质量为75 kD;将144枚重构胚移入16只同步发情的山羊输卵管中,在移植后的30 d、60 d和90 d的妊娠率分别为87.5%、81.3%和62.5%;最终3只受体妊娠足月,产下3只克隆羊,克隆效率为2.1%,PCR-RFLP分析表明克隆羊均来自供体羊细胞,但没有整合外源基因。结果表明,hLF转基因乳腺上皮细胞能分泌hLF;乳腺上皮细胞经转染、筛选和长期培养的条件下,能保持发育的全能性。     

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。     

外源基因转染导致山羊体细胞过快衰老与端粒缩短

在对山羊体细胞进行外源基因转染过程中,无论电击法或脂质体法所得到的细胞克隆都有细胞过快衰老的现象。山羊体细胞转基因后出现细胞体积增大、细胞核膨大并逐步分裂成多核、细胞质空泡化和吐核等衰老的表型特征。转基因后衰老细胞的染色体核型正常,但经细胞染色体端粒长度的Southern检测发现,转基因衰老细胞比原代胎儿成纤维细胞染色体端粒长度减少了2.56 kb,超出了正常传代40代的细胞的衰老速度,但转基因衰老细胞仍能支持核移植克隆胚胎的早期发育。     

山羊品种间体细胞核移植研究

萨能奶山羊是著名的奶用山羊品种,波尔山羊则是世界著名的肉用山羊品种.为了研究波尔山羊体细胞在奶山羊卵母细胞中的去分化,我们将成年波尔山羊的颗粒细胞或耳皮肤成纤维细胞作为供核细胞(试验组),移入奶山羊中Ⅱ期的去核卵母细胞透明带下,经电融合和离子霉素与6-二甲基氨基嘌呤(6-DMAP)激活,直接移入同期发情奶山羊输卵管或经体内培养,将发育的重构胚移人同期发情羊子宫内.妊娠早期作B超诊断,确立妊娠的观察至足月.同时将奶山羊的35日龄胎儿成纤维细胞作供核细胞(对照组),按试验组同样方法处理,将重构胚直接移入同期发情的奶山羊输卵管内.结果试验组,波尔羊颗粒粒细胞与耳皮肤成纤维细胞的融合率分别为78.2%(115/147)、57.4%(116/202),重构胚卵裂率为85.8%(115/134),桑椹胚、囊胚的发育率38.8%(52/134),早期妊娠三头,分别于妊娠40、60、60日龄终止妊娠.对照组,融合率为89.5%(136/152),早期妊娠率为42.9%(6/14),四头受体足月分娩,产四头公羊羔,其中三头存活,一头分娩时死于肺不扩张,并体重过大,显示胎儿过大综合症.经基因型鉴定证实,这四头克隆羔羊均源于同一胎儿成纤维细胞系.以上结果表明,波尔羊体细胞核在奶山羊卵母细胞中能够去分化,并维持一定程度的发育.     

山羊品种间体细胞核移植研究

萨能奶山羊是著名的奶用山羊品种,波尔山羊则是世界著名的肉用山羊品种。为了研究波尔山羊体细胞在奶山羊卵母细胞中的去分化,我们针成年波尔山羊的颗粒细胞或耳皮肤成纤维细胞作为供核细胞（试验组）,移入奶山羊中Ⅱ期的去核卵母细胞透明带下,经电融合和离子霉素与6－二甲基氨基嘌呤－DMAP）激活,直接移入同期发情奶山羊输卵管或经体内培养,将发育的重构胚移入同期发情羊子宫内。妊娠早期作B超诊断,确立妊娠的观察至足月。同时将奶山羊的35日龄胎儿成纤维细胞作供核细胞（对照组）,按试验组同样方法处理,将重构胚直接移入同期发情的奶山羊输卵管内。结果：试验组,波尔羊颗粒粒细胞与耳皮肤成纤维2细胞的融合率分别为78．2％（115／147）,57．4％（116／202）,重构胚卵裂率为85．8％（115／134）,桑椹胚,囊胚的发育率38．8％（52／134）,早期妊娠三头,分别于妊娠40,60,60日龄终止妊娠。对照组,融合率为89．5％（136／152）,早期妊娠率为42．9％（6／14）,四头受体足月分娩,产四头公羊羔,其中三头存活,一头分娩时死于肺不扩张,并体重过大,显示胎儿过大综合症。经基因型鉴定证实,这四头克隆羔羊均源于同一胎儿成纤维细胞系。以上结果表明,波尔羊体细胞核在奶山羊卵母细胞中能够去分化,并维持一定程度的发育。     

敲除山羊胎儿成纤维细胞中的抗体重链基因

在针对大动物的精确基因修饰研究中,基于体细胞的同源重组是唯一可行与有效的方法．其中,沉默基因位点的重组尤为困难．为获得抗体基因功能缺失的山羊用于人源化抗体的研究,通过体细胞同源重组技术,首次成功地获得了抗体基因敲除的山羊胎儿成纤维细胞株,该细胞株可用于体细胞克隆制备抗体基因功能缺失的转基因山羊．以35日龄的山羊胎儿成纤维细胞（GEF88）基因组DNA为模板,扩增山羊抗体重链J-Cμ基因作为同源臂,构建了同基因型的正负筛选打靶载体GTIgH．将此打靶载体经电穿孔的方法转染GEF88细胞,并通过0．8mg／L的嘌呤霉素进行药物筛选,获得了362个抗性细胞克隆,PCR、测序及DNA印迹鉴定结果显示,其中的GT211抗性细胞克隆为中靶细胞,该细胞克隆中的抗体重链基因的一条等位基因已被成功敲除．     

以不同类型的转基因细胞为核供体生产牛的转基因克隆胚胎

利用所构建的含新霉素抗性(Neo^r)基因和绿色荧光蛋白(GFP)基因的双标记选择载体, 通过电穿孔的方法, 分别转染了牛胎儿成纤维细胞、胎儿输卵管上皮细胞、胎儿卵巢上皮细胞、颗粒细胞, 经过800 μg/mL的G418筛选14 d后, 均获得了阳性细胞株. 分别以未转基因牛颗粒细胞和4种细胞系的转基因细胞为核供体, 进行了牛的体细胞核移植. 结果表明: (ⅰ) 转基因与未转基因牛颗粒细胞的重组胚的囊胚发育率(44.6% vs 42.8%)、移植妊娠率(19% vs 25%)差异不显著(P＞0.05); (ⅱ) 比较4种类型转基因细胞的重组胚的囊胚发育率, 发现胎儿输卵管上皮细胞(49.1%)和颗粒细胞(44.6%)最高, 牛胎儿成纤维细胞(37.2%)次之, 胎儿卵巢上皮细胞的重组胚囊胚发育率(22.5%)最低, 三者之间差异显著(P＜0.05). 以上结果显示, 供体细胞的转基因与否对牛克隆胚胎的体外和体内早期发育影响不明显; 通过体细胞核移植技术, 牛胎儿输卵管上皮细胞和颗粒细胞可以有效地生产牛转基因囊胚, 并且绿色荧光蛋白作为一种无毒性作用的筛选标记, 可用于转基因胚胎的筛选.     

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%).     

猪体细胞核移植重构胚的体外发育(英文)

以卵丘细胞为核供体细胞组成重构胚 ,卵裂率达到 5 6.7% ,发育至桑椹胚率达到1 1 .7% ,囊胚率为 6.7% ,显著高于成纤维细胞重构胚 (P <0 .0 5 )。本文还研究了卵母细胞的采集方法、激活程序和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至G0 G1 期 ,抽吸法 解剖法采集卵母细胞 ,体外培养 3 3～ 44h ,将卵丘细胞放至去核卵母细胞的卵周隙中 ,重构胚以钙离子载体A2 3 81 7或电脉冲结合 6 DMAP激活处理 ,体外培养 6d。研究表明 ,卵母细胞采集方法、激活液中细胞松弛素 (CB)、激活程序并不影响重构胚的发育 (以卵龄 44h的卵母细胞为受体 ) ;而以电脉冲结合 6 DMAP激活处理能提高重构胚发育能力 (以卵龄 3 3h的卵母细胞为受体 ) (P <0 .0 5 )。本研究显示 ,以电脉冲结合 6 DMAP激活卵丘细胞重构胚 ,体外能发育至囊胚     

影响猪体细胞核移植重构胚体外发育的若干因素

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56．7％,发育至桑椹胚达11．7％、孵化囊胚率为6．7％,显著高于成纤维细胞组成的重构胚（P＜0．05）。我们研究了卵母细胞的采集方法,激活方法和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至G0或G1期,抽吸法／解剖法采集卵母细胞,体外培养33或44h,将卵丘细胞置于去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电泳冲结合6－DMAP激活处理,体外培养6天,结果表明,卵 母细胞采集方法、激活液中细胞松弛素（CB）并不影响重构胚的发育（以卵龄44h的卵母细胞为受体）;而以电脉冲结合6－DMAP激活处理能提高重构胚发育能力（以卵龄33h的卵母细胞为受体）（P＜0．05）。本研究显示,以电脉冲结合6－DMAP激活卵丘细胞重构胚,能在体外发育至囊胚。     

影响猪体细胞核移植重构胚体外发育的若干因素

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56.7%,发育至桑椹胚达11.7%、孵化囊胚率为6.7%,显著高于成纤维细胞组成的重构胚(p<0.05)。我们研究了卵母细胞的采集方法,激活方法和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至GO或G1期,抽吸法/解剖法采集卵母细胞,体外培养33或44 h,将卵丘细胞置于去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电脉冲结合6-DMAP激活处理,体外培养6天,结果表明,卵母细胞采集方法、激活液中细胞松弛素(CB)并不影响重构胚的发育(以卵龄44h的卵母细胞为受体);而以电脉冲结合6-DMAP激活处理能提高重构胚发育能力(以卵龄33 h的卵母细胞为受体)(p<0.05)。本研究显示,以电脉冲结合6-DMAP激活卵丘细胞重构胚,能在体外发育至囊胚     

Production of cloned goats from enucleated oocytes injected with cumulus cell nuclei or fused with cumulus cells

This study was designed to produce cloned goats from cumulus cells. Cloning donor nuclei were from cumulus cells either freshly isolated or cultured in vitro. Enucleated oocytes were either injected with cumulus cell nuclei without piezo-driven manipulator (injection method) or fused with cumulus cells (fusion method). The survival rate of cloned embryos, obtained by injection, was higher than that derived from fusion (62.7 and 45.9%, respectively). Two cloned goats were derived by fusion with in vitro cultured cumulus cells without starvation, but died shortly after natural birth, from respiratory difficulties. Their birth weights (2.23 kg and 2.03 kg) were within the normal range (2.0-2.7 kg) and postmortem analysis revealed no morphological abnormalities. The third cloned goat, derived by injection of nuclei from freshly isolated cumulus cells, weighed 3.3 kg at birth, and was 37% overweight compared with the average weight of the same species. This goat is healthy and well as this paper is being prepared. Nested PCR-RFLP analysis confirmed that all the cloned goats were derived from the donor cells.     

Efficiency of cloned embryo production using different types of cell donor and electric fusion strengths in goats

The type of somatic cell used as a cell donor and the electric field strength (EFS) applied for membrane fusion of the reconstructed oocytes are the two important aspects that need to be standardized for somatic cell nuclear transfer (SCNT). In the present study two somatic cells types, namely fibroblast cell grown from ear tissue biopsies of Barbari female goats and cumulus cells were used as somatic donor cells. For fusion of oocyte reconstructed membranes following somatic cell transfer, a dc current of 3 electrical field strength (EFS), i.e., 1.0–1.5; 2.0–2.5; 3 and above 3, were applied. When cumulus cells were used as a nuclear donor, a maximum fusion rate of (55.4 ± 3.9%) was obtained by applying 2.0–2.5 kV/cm dc current. The fusion rate obtained was significantly (P < 0.05) higher than all the other EFSs treatments of cumulus, as well as fibroblast cell types. The maximum fusion rate (31.9 ± 2.4%) for the fibroblast cell line was observed when an EFS of 2.0–2.5 kV/cm was applied. It could be concluded that the difference in membrane surface properties between the cumulus and fibroblast cell may contribute to the higher fusion rate obtained in cumulus cells for cloned embryo production.     

Cloned mice derived from somatic cell nuclei

In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum.     

Cloned goats (Capra hircus) from adult ear cells

The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the MⅡchromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female Jining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5 μmol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no significant differences in development in vitro between the cloned embryos derived from donor cells precooled at 4℃ for 24 h and nonprecooled donor cells. The cleavage rates, 4-cell development, and blastocyst development of reconstructed embryos were 72.48% (79/109), 53.16% (42/79), and 19.05% (8/42) in precooled group; 68.5% (211/308), 59.72% (126/211), and 17.46% (22/126) in nonprecooled group, respectively. Eighteen cloned 4-cell embryos derived from precooled donor cells were transferred and one cloned kid was born. Eighty-four cloned 4-cell embryos derived from nonprecooled donor cells were transferred and no offspring were produced. Of 18 cloned morale from nonprecooled donor cells transferred, one kid was born. The results of microsatellite DNA analyses indicated that the two cloned kids were from the same donor fibroblast cell line derived from an adult goat ear skin.     

Cloned goats (Capra hircus) from adult ear cells

The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the MII chromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female dining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5μ mol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no signifi     

Generation of cloned goats (Capra hircus) from transfected foetal fibroblast cells, the effect of donor cell cycle.

The neomycin-resistant gene (neo(r)) is probably the most commonly used selectable marker gene in gene targeting and gene transfection research. In this study, the neo(r) gene construct was introduced into in vitro cultured goat foetal fibroblast cells (IV-5), and the cells were selected with 900 microg/ml G418. The G418-resistant colonies were analysed by neo-specific PCR, karyotyping and anti-intermediate filament proteins antibody (anti-vimentin) staining. Cell cycle analysis of the neo(r) positive foetal fibroblast cell colony (IV-5.1) cultured in a variety of cell cycle-arresting medium indicated that 74.2% of cells cultured in serum-deprived medium for 3 days and 71.7% of cells grown to confluence were at G0/G1 stage of cell cycle, respectively, in comparison to 61.6% of cells in normal culture (cycling) medium. Nocodazole treatment for 17 hr in vitro culture could increase the number of cells at G2/M stage of cell cycle from 20.3% (in cycling medium) to 39.7%. In total, one early pregnancy was observed by B ultra-sound scanning in a surrogate transferred with cloned embryos from IV-5.1 cells at M stage (cells were cultured in nocodazole medium). Seven cloned goats, including two that miscarried at a late stage, were derived from the IV-5.1 cell clone cultured in starved medium (G0). Indeed, one surrogate receiving three blastocysts reconstituted from the starved donor cells, gave birth to three live cloned goats, all of which are healthy and doing well. PCR, Southern blot and G418 resistance in vitro of fibroblast cells from cloned goats confirmed that all cloned goats are positive for neo(r) transgene. This study demonstrates that a foreign gene, such as the neo-resistant gene, can be introduced into goat foetal fibroblast cells, and that the resulting transgenic cells are capable of being cloned to produce 100% transgenic animals.