电穿孔法转染293T细胞条件的优化

为筛选电穿孔转染人胚肾293T细胞的最优条件,将增强型绿色荧光蛋白(EGFP)基因克隆至启动子p CMV前,获得的重组质粒在不同电压、质粒浓度和电击次数条件下电穿孔转染293T细胞,继而在倒置荧光显微镜下观察转染细胞,根据EGFP表达情况评价转染效率。结果表明400 V电压、45 mg质粒电穿孔转染293T细胞2次时转染效率达到44%,与脂质体转染效率(51%)无统计学差异。     

利用基于CRISPR-Cas9 的基因编辑技术对miR-29a在GT1-7 细胞中进 行基因敲除

目的:本文利用CRISPR-Cas9技术在GT1-7细胞中对miR-29a基因进行基因编辑,用于构建miR-29a基因敲除GT1-7细胞模型。方法:通过构建Cas9稳转的GT1-7细胞株并转染sgRNA质粒用于在靶向miR-29a基因区域引发突变。然后构建EGFP与sgRNA共表达质粒并转染Cas9稳转GT1-7细胞,利用流式细胞仪富集表达绿色荧光蛋白的阳性细胞和分选阳性单克隆细胞。最后利用实时荧光定量PCR(realtimefluorescencequantitativePCR)对富集细胞和单克隆细胞进行miR-29a表达量检测。结果:T7E1检测结果显示CRISPR-Cas9系统有效地在miR-29a基因区域引发了突变。荧光定量PCR结果显示,与对照组相比,富集后阳性细胞miR-29a的表达量整体下降了50%左右(P0.05)。此外,通过流式筛选获得了一个纯合miR-29a基因敲除细胞克隆,与对照组相比,其miR-29a的表达量下降了75%左右(P0.05)。结论:本文建立了一种有效编辑GT1-7细胞基因的方法,并采用该方法构建了miR-29a稳定敲除细胞模型。     

牛pAcGFP-FADD融合蛋白真核表达载体构建及在CHO-K1细胞中的表达

FADD是Fas/FasL系统的一个信号连接蛋白,通过传递凋亡信号,介导细胞凋亡.为了揭示FADD在牛卵泡发育过程中的调控作用,采用RT-PCR从牛卵巢组织中扩增FADD基因,将其cDNA终止密码子删除,采用定向克隆技术连接到带有水母绿色荧光蛋白(AcGFP)报告基因的真核表达载体pAcGFP-N1中,构建融合蛋白重组质粒,经BglⅡ/EcoR Ⅰ酶切、测序鉴定后,用脂质体介导质粒转染CHO-K1细胞,观察有无荧光的表达及用RT-PCR和Western blotting方法检测基因转录、表达情况.结果表明,成功克隆牛FADD基因,通过PCR方法在FADD阅读框两端引入了Bgl Ⅱ和EcoR Ⅰ克隆位点,并于起始位点前加入Kozak序列,成功构建pAcGFP- bFADD融合蛋白真核表达载体,重组质粒转染CHO-K1 24 h后在荧光显微镜下观察到绿色荧光,转染效率可达65%,通过RT-PCR扩增出654 bp的转录产物,并用Western blotting检测到51.4 kD目的蛋白的表迭.     

基于流式细胞术分选的高效表达外源基因细胞的筛选

目的：以增强型绿色荧光蛋白（EGFP）作为报告基因,用流式细胞术筛选高表达EGFP的细胞,从而获得外源基因高效表达细胞株。方法：构建在EGFPC端编码区融合新霉素（neomycin）抗性基因的融合基因EGFP-Neomycin,将其插入pcDNA3.1（＋）载体,构建EGFP-Neomycin融合基因表达载体pcDNAEN,转染CHO-K1细胞,G418加压筛选和倒置荧光显微镜观察证实所表达的EGFP-Neomycin融合蛋白具有新霉素抗性和激发EGFP荧光双功能;将编码组织型纤溶酶原激活剂（tPA）的cDNA插入pcDNAEN中CMV启动子下游,构建表达tPA的表达载体pcDNAEN/tPA。结果：流式细胞术分析和tPA纤维蛋白溶解活性测定表明,pcDNAEN/tPA转染CHO-K1细胞的EGFP相对荧光强度（RFT）的自然对数值与tPA表达水平呈明显的直线相关关系,相关系数为0.983;比较部分未经流式细胞仪分选的pcDNAEN/tPA转染阳性细胞克隆和RFT分布在100～1000的pcDNAEN/tPA转染阳性细胞克隆的tPA表达水平,经流式细胞术分选获得的细胞克隆的tPA平均表达水平和最高表达水平分别是未经分选获得的细胞克隆的3.9倍和4.1倍。结论：构建的EGFP-Neomycin融合基因具有双功能,建立了利用流式细胞术筛选外源基因高效表达物细胞株的方法。     

绿色荧光蛋白标记的D-氨基酸氧化酶基因在人宫颈癌细胞中的表达研究

为了探讨绿色荧光蛋白标记的红色酵母D 氨基酸氧化酶 (DAAO)基因在人宫颈癌细胞 (HeLa细胞 )中的表达及其功能 ,采用基因重组技术构建了含有CMV启动子和EGFP、DAAO基因开放阅读框 (ORF)的真核表达载体 pIRES DAAO。脂质体法转染HeLa细胞 ,荧光显微镜下观察转染细胞中绿色荧光蛋白的表达 ,流式细胞术分析转染效率并筛选荧光阳性细胞 ,命名为HeLa D。以不同浓度的前药D Ala处理HeLa D细胞 ,MTT法检测细胞存活率。结果显示 ,荧光显微镜下可见绿色荧光蛋白在HeLa D细胞中表达 ,流式细胞术成功筛选出HeLa D细胞。前药D Ala能明显杀伤HeLa D细胞。结果表明 ,EGFP可作为报告基因快速筛选DAAO表达载体转染的细胞 ,DAAO/D Ala自杀基因系统可进一步用于肿瘤的基因治疗研究     

ZNF268基因启动子区的克隆及功能分析

锌指基因家族是人体中最大的基因家族,它参与细胞分化、胚胎发育,并与许多疾病的发生相关。人类ZNF268基因是一个在人胚肝中特异性表达的C2H2型锌指基因,并可能在人的早期肝脏发育中起重要作用。为了研究ZNF268基因表达调控的分子机制,以正常人总基因组为模板PCR扩增了ZNF268基因的5′调控区2533bp片段,并将此片段插入启动子缺失的EGFP(增强型绿色荧光蛋白)载体构建了重组质粒pZNF268—EGFP。用脂质体介导的方法将pZNF268—EGFP转染NIH／3T3、COS7、K562、HeLa4个细胞系。在激光共聚焦显微镜下观察绿色荧光的表达,发现在每个细胞系中,转染了重组质粒pZNF268—EGFP的细胞均有荧光表达,但其起始表达时间均晚于转染了阳性对照质粒pEGFP—C1的细胞且荧光较弱。这表明ZNF268基因的5′调控区2．5kb片段是一个有功能的启动子,但该启动子与CMV启动子相比活性较弱。选择易培养的HeLa细胞系用于缺失研究。将一系列5′端—2456bp至—20bp缺失、3′端均为 77bp的缺失片段插入启动子缺失的CAT(氯酶素酰胺转移酶)载体构建了一系列重组质粒。将这些重组质粒转染HeLa细胞系进行缺失分析,并通过共转染pCMV—Sport—βgal质粒校正转染效率。结果表明,ZNF268启动子—2456～—1639bp区域可能含有正调控元件,—1244～—1013bp和—525～—156bp区域可能含有负调控元件,ZNF268启动子激活转录的一个重要区域位于—156～—20bp。     

抗病毒基因MxA真核表达载体的构建及在鸡细胞中的表达

将人抗病毒蛋白基因MxA与携带增强型绿色荧光蛋白(EGFP)基因的真核表达质粒重组后构建MxA基因真核表达载体pEGFP-C1-MxA.经PCR和酶切方法鉴定后,重组质粒在脂质体介导下转染鸡成纤维细胞和睾丸组织原代细胞,通过荧光观察,RT-PCR及细胞免疫组化检测目的基因的表达.结果表明,MxA基因片段已经被克隆到pEGFP-C1表达载体,成功构建了MxA基因真核表达载体pEGFP-C1-MxA.经该重组质粒转染后的鸡细胞的胞质中呈现颗粒状分布的绿色荧光,RT-PCR扩增出EGFP和MxA基因的特异性片断,免疫组化结果显示EGFP报告基因在细胞内的阳性表达,并表现出MxA的表达特征,间接证明了MxA可在鸡细胞中表达.MxA基因真核表达载体的成功构建以及在鸡细胞中的表达为进一步研究MxA基因在抗禽病毒性疾病中的应用打下了基础.     

小鼠DLL1真核表达载体的构建及其在肿瘤细胞中的表达

目的：构建带绿色荧光蛋白的小鼠DLL1全长基因真核表达载体,并在肿瘤细胞中表达。方法：利用PCR特异性引物扩增出DLL1基因全长,将克隆的基因片段插入带绿色荧光蛋白的真核表达载体pIRES2-EGFP质粒中。然后利用脂质体将重组质粒pIRES2-EGFP-DLL1转染进小鼠B16黑色素瘤细胞中,并通过G418筛选后选取生长良好、荧光强度高的三株单克隆进行mRNA水平DLL1表达的鉴定。结果：成功扩增小鼠DLL1的全长基因。克隆入质粒载体后,通过DNA序列测定证实其序列正确。将构建的pIRES2-EGFP-DLL1质粒转染小鼠B16黑色素瘤细胞,经过G418筛选和荧光显微镜观察后,挑选得到GFP阳性率90%以上的稳定转染细胞株。RT-PCR检测稳定转染细胞的mDLL1的表达显著增加,进一步证实了pIRES2-EGFP-DLL1的表达效能。结论：成功构建了小鼠DLL1基因的真核表达质粒,证实其在真核细胞B16中可以表达。     

小鼠DLL1真核表达载体的构建及其在肿瘤细胞中的表达

目的:构建带绿色荧光蛋白的小鼠DLL1全长基因真核表达载体,并在肿瘤细胞中表达。方法:利用PCR特异性引物扩增出DLL1基因全长,将克隆的基因片段插入带绿色荧光蛋白的真核表达载体pIRES2-EGFP质粒中。然后利用脂质体将重组质粒pIRES2-EGFP-DLL1转染进小鼠B16黑色素瘤细胞中,并通过G418筛选后选取生长良好、荧光强度高的三株单克隆进行mRNA水平DLL1表达的鉴定。结果:成功扩增小鼠DLL1的全长基因。克隆入质粒载体后,通过DNA序列测定证实其序列正确。将构建的pIRES2-EGFP-DLL1质粒转染小鼠B16黑色素瘤细胞,经过G418筛选和荧光显微镜观察后,挑选得到GFP阳性率90%以上的稳定转染细胞株。RT-PCR检测稳定转染细胞的mDLL1的表达显著增加,进一步证实了pIRES2-EGFP-DLL1的表达效能。结论:成功构建了小鼠DLL1基因的真核表达质粒,证实其在真核细胞B16中可以表达。     

TurboFect介导骨形态发生蛋白2 转染CHO 细胞的研究

目的:构建真核表达载体p IRES-EGFP-BMP-2,通过Turbo Fect转染得到表达BMP-2蛋白的CHO细胞系。方法:利用逆转录PCR方法扩增获得人的BMP-2基因c DNA,克隆入p MD18-T载体,经PCR、酶切和基因测序分析等方法鉴定重组质粒;将BMP-2连入p IRES-EGFP真核表达载体中,经限制性酶切和PCR扩增鉴定重组质粒。以壳聚糖和Turbo Fect分别作为基因载体转染CHO细胞,荧光显微镜检测分析转染结果;G418筛选富集转染阳性细胞。结果:成功的克隆得到了BMP-2基因,酶切鉴定成功构建了p IRES-EGFP-BMP-2质粒。与壳聚糖组相比,Turbo Fect用量为1:1时,细胞阳性率为(31.92±1.31)%,高于壳聚糖的细胞阳性率(6.33±1.53)%。目的基因与Turbo Fect比例为1:2时转染效率为(42.90±1.10)%高于1:1的(28.59±2.38)%和1:3的(37.52±2.14)%。细胞密度调节到5×103 cells/cm2阳性细胞率可达到(44.43±3.23)%。荧光检测可见荧光阳性细胞得到稳定传代。Western Blot检测可见BMP-2蛋白表达。结论:Turbo Fect成功的介导了p IRES-EGFP-BMP-2载体转染CHO细胞,建立了稳定表达BMP-2和EGFP的CHO细胞株。     

人胃癌Ha-ras基因及上游区片段甲基化对转化效率及表达的影响

 本文将克隆于pBR322的人胃癌Ha-ras基因(PGC6.6)和带有上游区片段的Ha-ras基因(PGC9.1)的CC~*GG位点甲基化后,转化NIH3T3细胞。发现pGC6.6甲基化与非甲基化对转化效率无明显影响,而pGC9.1甲基化后转化效率明显低于非甲基化pGC9.1者,甲基化/非甲基化pGC9.1的转化效率均明显高于甲基化/非甲基化pGC6.6者。本文又对人胃癌组织及癌旁组织DNA中Ha-ras基因的HpaⅡ、Msp Ⅰ限制性内切酶图谱作了比较,并同对比较了癌及癌旁组织中Ha-ras基因的mRNA水平,发现一例病人癌组织中Ha-ras基因的CC~*GG位点甲基化程度较癌旁组织中者低,且该例中Ha-ras基因表达水平在癌组织中明显地高。这些结果,结合我们以前的研究表明:在人胃Ha-ras癌基因上游区可能存在一增强子样作用的区域,对Ha-ras基因起调控作用。该上游区CC~*GG位点的甲基化能降低这种调控作用。仅Ha-ras结构基因的CC~*GG位点甲基化不足以明显影响其转化活性。在体内,Ha-ras基因甲基化水平降低可能与其达表水平升高以至诱发癌症有关。     

生长分化因子-9对牛卵丘细胞增殖的影响

研究生长分化因子-9（growth differentiation factor-9,GDF-9）对牛卵丘细胞增殖的影响。采用MTT法检测不同浓度GDF-9对卵丘细胞增殖的影响,结果表明,GDF-9能促进卵丘细胞的增殖,且GDF-9与卵丘细胞增殖效应存在浓度梯度关系;在卵丘细胞增殖过程中,FSH在一定程度上与GDF-9发挥协同作用。在GDF-9和FSH的作用下,去除卵母细胞的卵丘细胞复合体（oocytectomized cumulus cell complexes,OOX）也可以保持较好的发育形态。实时定量PCR结果表明,随着GDF-9浓度的增加,卵丘细胞扩展相关基因PTX3、HAS2及PTGS2的表达量也增加。总之,以上的研究结果表明,GDF-9可以促进卵丘细胞的增殖,对卵丘细胞功能的发挥起着重要的作用。     

Bovine cumulus cell expansion does not depend on the presence of an oocyte secreted factor

Communication between the oocyte and its somatic cells has been shown to be important in oocyte development. Here we examined how the oocyte may be involved in bovine cumulus cell expansion. Intact bovine cumulus oocyte complexes (COC) were obtained by puncturing antral follicles. From the intact COC, oocytectomised complexes (OOX) were produced by micro surgical removal of the oocyte. Clumps of cumulus cells (CC) were obtained by micro-dissection. Intact or OOX complexes or CC were matured in the presence of fetal calf serum and hFSH (6 mlU/ml) for 24 hr and the degree of expansion measured. The presence of the oocyte is not essential to allow bovine cumulus expansion to occur as expansion occurred in all groups. Murine OOX complexes from eCG primed 35–40-day-old C57BL6/CBA F₁ hybrids (known to require the presence of an oocyte secreted factor for cumulus expansion) were cultured with or without denuded bovine oocytes (1 oocyte/μl). Murine OOX complexes expanded only in the presence of denuded bovine oocytes. Thus some factor produced by bovine oocytes enabled expansion of murine OOX complexes. To determine whether the factor is secreted by bovine oocytes, murine OOX were cultured with or without media conditioned by bovine oocytes (1 oocyte/μl for 4 hr). Significant expansion of murine OOX occurred in media conditioned by bovine oocytes. This shows that the cumulus expansion enabling effect of bovine oocytes is released into the surrounding media. Media conditioned by bovine oocytes and then frozen for up to 1 month showed that the activity by the factor can withstand freezing. © 1995 wiley-Liss, Inc.     

Effect of the cumulus on in vitro fertilization of bovine matured oocytes

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO₂ in air and heparin.

In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.     

牛c-myc基因的克隆及其在皮肤成纤维细胞中的表达

为了构建包含牛c-myc基因编码序列的重组载体,以胎牛原始生殖嵴为材料,用RT-PCR方法克隆出牛c-myc 基因的编码序列,将其亚克隆至pMD19-T载体,再从酶切鉴定和测序正确的质粒上切下目的片段,定向克隆到pIRES2-AcGFP1-Nuc表达载体上,挑选序列正确的重组真核表达质粒转染牛皮肤成纤维细胞,用RT-PCR和Western blotting分别检测c-myc mRNA和蛋白的表达。结果表明,从胎牛原始生殖嵴中正确克隆了c-myc基因的全长编码序列,所构建的重组质粒能够在皮肤成纤维细胞中有效     

Effects of cumulus cell density during in vitro maturation of the developmental competence of bovine oocytes

To determine the role of cumulus cells in oocyte maturation, we carried out an investigation on the effects of addition of cumulus cells to the maturation medium on the developmental competence of corona-enclosed oocytes and oocytes denuded from their somatic cells. The addition of cumulus cell (1.6 x 10(6) cells/mL) improved the development of bovine corona-enclosed oocytes, however, addition of a similar number of cumulus cells as cumulus-oocyte-complexes (COCs, cumulus cell density: 4.2 x 10(6) cells/mL) had no effect on the development of oocytes denuded from their somatic cells. To determine if corona-enclosed oocytes can obtain developmental competence without the addition of extra cumulus cells, the effects of cell density during in vitro maturation on the developmental competence were studied. A density of 1.6 to 3.2 x 10(6) cumulus cells/mL was the most effective for in vitro maturation of oocytes with intact gap junctions. The effects of the medium conditioned by COCs on the developmental competence of oocytes was also examined. It was demonstrated that COC-conditioned medium improved the development of bovine oocytes to the blastocyst stage. These data suggest that the developmental competence of bovine oocytes surrounded with corona cells is supported in a cell density-dependent manner in the maturation medium. In addition, the data indicate that cumulus cells benefit bovine oocyte development either by secreting soluble factors which induce developmental competence or by removing an embryo development-suppressive component from the medium.     

Possible role of 5'AMP-activated protein kinase in the metformin-mediated arrest of bovine oocytes at the germinal vesicle stage during in vitro maturation

The 5'AMP-activated protein kinase (AMPK) activation is involved in the meiotic maturation of oocytes in the ovaries of mice and pigs. However, its effects on the oocyte appear to be species-specific. We investigated the patterns of AMPK and mitogen-activated protein kinases (MAPK3/1) phosphorylation during bovine in vitro maturation (IVM) and the effects of metformin, an AMPK activator, on oocyte maturation in cumulus-oocyte complexes (COCs) and denuded bovine oocytes (DOs). In bovine COCs, PRKAA Thr172 phosphorylation decreased, whereas MAPK3/1 phosphorylation increased in both oocytes and cumulus cells during IVM. Metformin (5 and 10 mM) arrested oocytes at the GV stage in COCs but not in DOs. In COCs, this arrest was associated with the inhibition of cumulus cell expansion, an increase in PRKAA Thr172 phosphorylation, and a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. However, the addition of compound C (10 muM), an inhibitor of AMPK, accelerated the initiation of the GV breakdown (GVBD) process without any alteration of MAPK3/1 phosphorylation in oocytes from bovine COCs. Metformin decreased AURKA and CCNB1 protein levels in oocytes. Moreover, after 1 h of IVM, metformin decreased RPS6 phosphorylation and increased EEF2 phosphorylation, suggesting that protein synthesis rates were lower in oocytes from metformin-treated COCs. Most oocytes were arrested after the GVBD stage following the treatment of COCs with the MEK inhibitor, U0126 (100 micromoles). Thus, in bovine COCs, metformin blocks meiotic progression at the GV stage, activates PRKAA, and inhibits MAPK3/1 phosphorylation in both the oocytes and cumulus cells during IVM. Moreover, cumulus cells were essential for the effects of metformin on bovine oocyte maturation, whereas MAPK3/1 phosphorylation was not.     

Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation

Developmental competence of oocytes is compromised if they originate from atretic follicles. Apoptosis is the underlying process of atresia. Apoptotic changes in follicular cells are thought to influence the outcome of IVF. The aim of this study was to investigate apoptosis in different compartments of single bovine follicles (follicular wall, granulosa and cumulus cells (CC)) in relation to COC morphology, and to determine whether the addition, in vitro, of exogenous follicular cells from atretic follicles to maturing cumulus oocyte complexes (COCs) influenced the development of oocytes.Antral follicles were dissected from bovine ovaries and opened to obtain COCs and free floating granulosa cells (GC). The COCs were classified according to morphology. Apoptosis was determined in cumulus and granulosa cells and in homogenates of the remaining follicular wall.For every morphological class of COCs, a large variability of apoptotic expression was found in all follicle compartments. Follicular wall apoptosis was not correlated to COC morphology or to the percentage of apoptotic granulosa or cumulus cells. In grade 1 (best morphology) COCs, the degree of apoptosis in granulosa cells was comparable to cumulus cell apoptosis (P<0.01). The overall expression of apoptosis in granulosa cells of follicles containing grade 3 COCs (median+/-median absolute deviation: 37.8+/-13.8%) was significantly higher (P<0.05) than in follicles with grade 1 (22.7+/-10.4%) or grade 2 COCs (20.0+/-17.0%). About 48.3% of grade 3 COCs possessed strongly apoptotic cumulus cells compared to 27.8 and 28.2% of grade 1 or grade 2 COCs, respectively. Nonapoptotic cumulus complexes were observed in grades 1 and 2 COCs only.Adding exogenous follicular cells from atretic follicles to bovine COCs (grades 1 and 2) during in vitro maturation (IVM) had no impact on fertilization, blastocyst formation or hatching after IVF. This is of particular practical relevance to embryo production after ovum pick up (OPU), as during this process, good quality COCs are cultured together with simultaneously collected slightly atretic COCs.     

Analysis of development-related gene expression in cloned bovine blastocysts with different developmental potential

The high incidence of abnormalities in cloned calves is a most serious problem for bovine somatic cell nuclear transfer (NT) technology. Because there is little information on the differences in mRNA expression in cloned blastocysts with donor cells of different sex and origin, we compared development-related gene expression in two types of cloned bovine blastocysts with different potentials to develop into normal calves, a female adult cumulus cell line (high potential to develop into live calves) and a male fibroblast cell line (low potential to develop into live calves) to examine the correlation between the normality of cloned calves and blastocyst mRNA expression patterns. We analyzed 12 genes involved in apoptosis, growth factor signaling, metabolism, and DNA methylation in blastocysts originating from two types of donor cells and in vitro-fertilized blastocysts using quantitative real-time polymerase chain reaction. Expression of the pro-apoptotic Bax gene and anti-apoptotic Bcl-2 and Glut-1 genes in fibroblast-derived blastocysts was significantly higher than in cumulus cell-derived and in vitro-fertilized blastocysts. The high Bcl-2 and Glut-1 gene expression suggests that some embryonic cells with damaged DNA in fibroblast-derived blastocysts are not removed, and their descendants later manifest abnormal placenta or fetus formation. Transfer of pre-selected cloned blastocysts into recipients is required, however, to determine whether the expression pattern of these apoptosis-related genes reflects differences in the potential to develop into normal calves.