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   总被引：2，自引：1，他引：1  

宋军  王振坤  孔庆然  赵祥杰  刘丽清  田江天  郑重  孙爽  尹智  刘忠华 《东北农业大学学报》2008,39(3):64-69

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。  相似文献   

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

LAN Jie  SONG Yong-li  HUA Song  LIU Yong-gang  LIU Jun  ZHANG Hai-lin  ZHANG Yong 《中国农业科学(英文版)》2010,9(7):1035-1040

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.  相似文献   

哺乳动物体细胞克隆技术应用的研究进展     

李继连  王丽  胡满 《中国畜牧兽医》2011,38(6):98-102

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。  相似文献   

哺乳类动物胚胎干细胞多能性和全能性研究进展   总被引：4，自引：1，他引：3  

安立龙  杨奇  雷安民  窦忠英 《西北农业学报》1999,8(2):113-116

交ＥＳ细胞分离与克隆技术和分子生物学技术相结合就可将新的遗传物质导入家畜的生殖腺细胞。通过囊胚注入胚胎干细胞产生嵌合体小鼠表明对胚胎干细胞进行遗传操作是对物生殖细胞进行遗传改造的有效方法之一。  相似文献   

牛繁殖技术的新进展     

张晓华  罗光彬 《现代畜牧兽医》2005,(1):44-46

动物繁殖技术是生物技术的重要分支。繁殖技术从20世纪50年代人工授精技术到20世纪70年代胚胎移植技术的应用,似乎才真正开始动物繁殖技术的新纪元。牛的繁殖技术在过去的50年里取得了显著进展,这是一个以人工授精起始的连续过程。随着体外受精、精子分离和核移植（克隆）等技术的发展,这些连续性的繁殖技术将是目前和未来提高生产力的强有力手段。  相似文献   

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

XU Guo-qi  HUA Zai-dong  XU Sheng-cheng  ZHENG Xin-min 《中国畜牧兽医》2016,43(3):754-760

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.  相似文献   

牛体外受精胚胎细胞核移植的实验研究     

李子义  卢圣忠 《中国兽医学报》1996,16(1):28-32

将采自肉联厂屠宰车间的牛卵巢中的卵母细胞置于ＴＣＭ－１９９＋１０％ＥＣＳ（０ｄ）＋ＦＳＨ（１万ＩＵ／Ｌ）＋ＬＨ（５ｍｇ／Ｌ）中培养成熟，用含０．３％透明质酸酶的消化液将卵母细胞周围的卵丘细胞去掉，并以７．５ｍｇ／Ｌ的ＣＢ液处理后，用微吸管吸去透明带内的第一极体及极体下面的部分卵母细胞质，然后将经体外受精并发育至８～１６细胞期胚胎的卵裂球注入去核卵母细胞的卵周隙中，再将移核胚置于电融合槽内进行融合（电场强度为１２００Ｖ／ｃｍ，持续时间为８０μｓ，１次脉冲刺激）。将融合的胚胎移入生长有单层贴壁颗粒细胞的发育培养液（ＴＣＭ－１９９＋１０％牛血清）中培养，观察移核胚的融合率及发育率，部分去核卵母细胞经染色后观察去核率。结果表明：（１）移核胚的融合率为８６．９％（５３／６１）；（２）发育至２～８细胞期的胚胎占培养的移核胚胎总数的２１．３％（１０／４７）；（３）卵母细胞的去核率为６８．２％（４５／６６）。  相似文献   

大鼠成纤维细胞同步处理的流式细胞仪分析   总被引：3，自引：0，他引：3  

刘志春  李六金  杨贵忠  冯秀亮  谢松涛  朱华萍  张生勇 《中国牛业科学》2002,28(6):16-18

目的 :研究 SD大鼠成纤维细胞同步处理后的各细胞周期的变化 ,探讨供体细胞的细胞周期对核移植胚胎发育的影响。方法 :分离培养 SD大鼠皮肤成纤维细胞 ,传 3～ 5代 ,分别经血清饥饿 2～ 6d和 1 0 mg/ ml nocodazole处理 2 4 h,流式细胞仪分析处理组和对照组的细胞周期百分比。结果 :血清饥饿、nocodazole处理及对照组的 G0 + G1期成纤维细胞的百分比 ( % )分别为 74.5± 4.4、49.5± 4.65和 5 0 .1 2± 3.9;G2 / M期细胞的百分比 ( % )分别为 :1 9.2 5± 4.2、2 5 .98± 2 .7及 45 .9± 5 .2 1。结论 :血清饥饿能较好地使大鼠成纤维细胞同步于 G0 和 G1期 ;nocodazole处理则使细胞较大程度同步于 G2 和 M期。  相似文献   

哺乳动物的核移植（综述）     

张继慈 《上海畜牧兽医通讯》1990,(3):43-44

哺乳动物卵细胞的核移植仅有几年的历史,但已在小鼠及牛、羊、猪等动物中获得了核移植的后代,取得了可喜的进展.下面就核移植法、融合法、研究成果与应用等作一介绍.一、核移植法据报道,迄今使用过的核移植法有三种,一种是 Illmensee 和 Hoppe(1981)的注入法,一种是McGrath 和 Solter(1983)的非破裂法,另一种是 Tsunoda 等(1988)的切开透明带法.Illmen-see 和 Hoppe 的方法是将小鼠囊胚(笫5天)的内细胞团和第7天胚胎外胚层的核注入除去雌、雄原核的受精卵细胞质内,然后移植到假孕小鼠的输卵管中,成功产下3只仔鼠.此后,他们用孤雌生殖卵的囊胚内细胞团的核进行了同样的移植,从重组  相似文献   

影响核移植胚胎存活率的因素     

Smit.  LC 岳奎忠 《黑龙江畜牧科技》1995,(1):62-64

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