利用体细胞核移植技术生产表达绿色荧光蛋白转基因猪胚胎的研究

试验对脂质体转染猪胎儿成纤维细胞的技术程序进行了筛选,以绿色荧光蛋白基因转染后的阳性细胞作为体细胞核移植的核供体,以体外成熟卵母细胞为核受体构建了绿色荧光蛋白转基因克隆猪胚胎,并对重构胚在体外发育情况以及绿色荧光蛋白表达情况进行了跟踪研究。结果显示,采用4.0μg.mL-1脂质体转染试剂,1.6μg.mL-1质粒DNA,转染6 h可以获得最佳转染效果,转染效率达4.48%;GFP转基因体细胞重构胚体外囊胚发育率为10%,GFP阳性胚胎率为48%。结果表明,脂质体转染试剂可以高效转染猪胎儿成纤维细胞,获得的阳性细胞具有支持猪全程发育的潜能。     

亮甲酚蓝对猪卵母细胞体外成熟及核移植胚胎发育能力的影响

试验旨在研究亮甲酚蓝(brilliant cresyl blue,BCB)染色对卵母细胞体外成熟及后期胚胎发育潜力的影响。本研究利用13、26、39 、52 μmol/L BCB对成熟培养前的卵丘－卵母细胞复合体(cumulus-oocyte-complexes,COCs)染色90 min,比较各组卵母细胞的着色率、成熟率及孤雌激活胚胎和核移植胚胎的发育情况。结果表明,随着BCB浓度的增加,COCs着色率依次增加(20.00%、46.39%、51.66%和59.03%),但猪卵母细胞体外成熟率逐渐降低(74.03%、72.16%、70.53%和48.61%);不同浓度BCB染色后所得BCB⁺组卵的成熟率均明显高于BCB^-组。试验结果发现,BCB浓度在26 μmol/L时,经染色的COCs既有较高的着色率,且不影响其体外成熟的效率。基于此,研究选取26 μmol/L BCB作为最佳浓度对猪卵母细胞进行染色筛选,然后进行体外培养、孤雌激活及核移植试验。结果显示,筛选的BCB⁺组卵母细胞的孤雌胚和核移植胚的卵裂率和囊胚率均显著高于BCB^-组(P<0.05),而与对照组间无显著差异(P>0.05)。胚胎移植试验挑选BCB⁺组中发育较好的1-2细胞期重组胚对5头代孕母猪进行了移植,其中2头怀孕,1头顺利产下了6头健康胎儿。综合以上试验结果表明,利用BCB染色可作为一种有效的方法筛选体外成熟质量较高的猪卵母细胞,同时提高胚胎体外生产效率。     

小白鼠颗粒细胞核移植操作问题的探讨

小白鼠颗粒细胞核移植技术是指将小白鼠的颗粒细胞,通过显微手术和细胞融合的方法,移植到一个去核卵母细胞中,构成重组胚胎(克隆胚胎),然后移植给受体,获得与核供体基因型相同的后代(克隆动物)的生物技术。目前,在核移植操作过程中还存在着许多问题,影响了核移植的效率。1供体和受体细胞的获取1.1超数排卵超数排卵是核移植中获取供体和受体细胞的关键技术。小白鼠的超数排卵常用孕马血清促性腺激素(PMSG)和人绒毛膜促性腺激素(hCG)[1]。由于小白鼠的个体差异以及不同的饲养环境,注射激素的剂量有所不同,剂量过大或过小都有可能影响超数排…     

哺乳动物体细胞克隆技术应用的研究进展

哺乳动物体细胞克隆技术是一项高新的生物技术,这一技术不仅为许多学科发展带来了机遇,它的应用将给畜牧业生产、转基因动物和医学带来了革命性的变化。作者就这几个方面的应用做一简要综述。     

cDNA Cloning of Goat DNA Methyltransferase 1,Screening of shRNA Vectors and Influences to Development of Nuclear Transfer Embryos

This study was designed to clone cDNA of goat DNA methyltransferase 1（DNMT1） gene,to screen an effective shRNAproducing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells.In this study,PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions.Next,3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector,acquiring 3 new vectors,respectively termed pRNAD1,pRNAD2 and pRNAD3.Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing.Finally,the developmental rates of nuclear transfer（NT） embryos generated using donor cells with and without the effective shRNA vector respectively,as well as in vitro fertilization（IVF） embryos were observed and recorded.The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned（GenBank no.FJ617538）.Furthermore,an effective interfering shRNA（pRNAD2） was obtained,with its interference effect being 47.88%.Finally,NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls（P 〈 0.05）,reaching the similar rates to IVF embryos（P 〉 0.05）.In conclusion,goat DNA methyltransferase 1 gene cDNA was cloned and sequenced,an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved,which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.     

Effect of BCB Staining on in vitro Maturation and Development of Nuclear Transfer Embryo of Pig Oocyte

This experiment aimed to study the effect of brilliant cresyl blue (BCB) on in vitro maturation of pig oocytes and the developmental capacity of pig SCNT embryos.The cumulus-oocyte complexes (COCs) were stained with different concentrations of BCB (13,26,39 and 52 μmol/L) for 90 min,and then we divided the COCs into BCB⁺ and BCB^- for in vitro culture 42 to 44 h.The results showed that,with the concentration of BCB increased,the staining rate (20.00%,46.39%,51.66% and 59.03%) raised gradually while the maturation rate of oocytes (74.03%,72.16%,70.53% and 48.61%) reduced,the percentages of oocytes staining by 26 μmol/L BCB for 90 min were higher than that of other groups in staining rate and maturation rate.However,the nuclear maturation rate of BCB⁺ groups were higher than that of BCB^- group.Therefore,26 μmol/L BCB was selected as the most effective concentration dying the oocytes (BCB⁺),which were used as parthenogenetic activation and nuclear transfer embryos.The cleavage and blastocyst rates of parthenogenetic activation and SCNT embryos in BCB⁺ group were significantly higher than that of BCB^- group (P<0.05),but there were no significant differences between the cleavage and blastocyst rates in the groups of BCB⁺ and control (P>0.05).Reconstructed embryos derived from the COCs stained with BCB were transferred to five surrogates,and six cloned piglets were obtained from one of the two pregnant pigs.These results showed that COCs stained with BCB was an effective method to select high-quality oocytes,which could improve the efficiency of in vitro embryo production.     

CRISPR/Cas9系统介导的人乳铁蛋白基因在山羊β-乳球蛋白基因座定点敲入

为研究山羊乳蛋白基因编辑，实现羊乳“人源化”改造。利用CRISPR/Cas9系统敲除山羊乳中致敏原β-乳球蛋白（BLG）基因，同时在BLG基因座定点敲入人乳铁蛋白（hLF）基因。针对山羊BLG基因第一外显子设计构建sgBLG/Cas9表达载体经电转染山羊胎儿成纤维细胞验证其编辑活性；将打靶载体BLC14与sgBLG/Cas9载体共转染山羊胎儿成纤维细胞，经筛选检测获得BLG~-/hLF~+打靶细胞株作为供核细胞用于山羊体细胞核移植；通过剖宫产获取克隆胎儿并验证其中靶情况。结果表明：sgBLG/Cas9编辑山羊BLG位点致突变率约为30%～35%；共筛选72株药物抗性细胞株，其中有37株为基因打靶细胞，打靶效率为51.4%(37/72）；挑取形态较好的7株打靶细胞用于核移植，共制备416枚重构胚，移植30只受体山羊；30-35日龄B超诊断有13只受孕，妊娠率为43.3%(13/30）；剖宫产获取3只35日龄克隆胎儿均验证为BLG~-/hLF~+基因型，并建立了BLG~-/hLF~+靶向性修饰细胞系。因此，利用CRISPR/Cas9系统可以获得BLG基因座定点打靶hLF基因的转基因克隆山羊，该研究为培育羊乳中低致敏原和富含hLF功能营养成分的转基因山羊新品系提供了科学依据。     

不同受体胞质对牛体细胞核移植效果的影响

通过研究不同受体胞质对核移植效果的影响,结果发现:部分体外成熟16~18 h的牛卵母细胞,其细胞核已完成成熟,胞质可直接用于核移植而不需进行激活处理,直接进行核移植囊胚率达7.7%;体外成熟培养(IVM)30 h的TⅡ期受体胞质较IVM 20~22 h激活的TⅡ期胞质核移植效果差,不适宜作受体胞质;MⅡ期胞质核移植效果优于TⅡ期胞质,两组囊胚率分别为11.7%和5.2%(P<0.05)。     

供体核种类对水牛核移植效果的影响

采用电融合法和直接注射法进行核移植,以比较不同来源和不同类型水牛供体细胞的核移植效果。当用电融合法进行核移植时,成体耳部成纤维细胞的融合率(46.0%)、卵裂率(53.9%)和囊胚发育率(10.9%),均显著低于胎儿成纤维细胞(64.5%,70.1%和21.6%)、胎儿皮肤细胞(71.5%,70.8%和22.1%)以及卵巢颗粒细胞(88.2%,79.1%和25.5%);后三种细胞间的卵裂率和囊胚发育率无显著差异,但卵巢颗粒细胞的融合率显著高于胎儿成纤维细胞和胎儿皮肤细胞(P<0.05)。当用直接注射法进行核移植时,胎儿皮肤细胞注核后的存活率(78.2%)、卵裂率(41.6%)均显著低于成体耳部成纤维细胞(88.6%和57.4%)和胎儿成纤维细胞(85.8%和65.0%)。胎儿成纤维细胞注核后的卵裂率和囊胚发育率(23.3%)均显著高于胎儿皮肤细胞(10.1%)和成体耳部成纤维细胞(12.0%)。结果表明:(1)采用直接注核法进行核移植时,胎儿成纤维细胞作为核供体较为合适,而采用电融合法进行核移植时,用颗粒细胞作核供体更为适宜;(2)胎儿成纤维细胞的核移植效果好于成体成纤维细胞。     

核移植技术在建立乳腺生物反应器中的应用

乳腺生物反应器的开发为制药业带来了生机,但目前采用的动物基因转移的方法,由于其各自固有的局限性,未能使得乳腺生物反应器的研究取得长足的进步。而核移植技术的发展为乳腺生物反应器的开发注入了活力。因此,笔者综合近年来国内外的文献,提出了核移植中供体细胞的选择以及处理方法,同时也从分子水平阐述了乳腺的一些调控机制,为核移植技术在乳腺生物反应器中的应用奠定了基础。