利用体细胞核移植技术制作人胰岛素原转基因牛 （英文）
Human Pro-insulin Transgenic Calf Derived from Somatic Cell Nuclear Transfer
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摘要: 通过体细胞核移植技术制作了人胰岛素原转基因牛。在CMV启动子指导下以内部核糖体进入位点序列（IRES）连接的新霉素抗性基因和绿色荧光蛋白基因组成了双重标记基因的筛选系统，用于转基因细胞的富集以及细胞和植入前胚胎的筛选。转基因通过电穿孔的方法（900 V/cm, 5 ms）转入体外培养的牛胎儿成纤维细胞，基因转染细胞在添加G418(800 μg/mL) 的培养基中培养10天以富集转基因细胞。选择表达绿色荧光蛋白的转基因细胞作为核供体进行体细胞核移植，重构胚经体外培养至囊胚阶段，选择表达绿色荧光蛋白的囊胚进行胚胎移植。为比较基因转染以及供体细胞所处周期对转基因细胞核移植胚胎发育的影响，用作核移植供体的转基因细胞或非转基因细胞先饥饿培养2—4天(0.5% FBS)，然后恢复培养(10% FBS)10?h使细胞同步化于G1期，以正常培养的细胞作为对照进行核移植。 结果表明，转基因细胞作为核供体得到的核移植胚胎的体外囊胚发育率低于以非转基因细胞为核供体的对照组(23.2% VS 35.2%, P<0.05)；转基因细胞周期同步化处理与否对其克隆胚囊胚发育率无显著影响（23.2% VS 18.9%, P>0.05）。胚胎移植后2个月直肠检查发现7头受体牛（每头移植2—4枚胚胎）中有一头妊娠，并最终发育足月产下一头小牛。聚合酶链反应（PCR）检测和DNA测序分析表明其为转人胰岛素原基因的转基因克隆牛。Abstract: The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer (SCNT). A double selection system, Neomycin resistance (Neor) gene and enhanced green fluorescent protein (EGFP) gene linked through an inner ibosomal entry site (IRES) sequence directed by a Cytomegalovirus(CMV) promoter, was used for enrichment and selection of the transgenic cells and preimplantation embryos. Transgenes were introduced into bovine fetal fibroblast cells (BFF) cultured in vitro through electroporation (900 V/cm, 5 ms). Transgenic bovine fibroblast cells (TBF) were enriched through addition of G418 in culture medium (800 μg/mL). Before being used as a nuclear donor, the TBF cells were either cultured in normal conditions (10% FBS) or treated with serum starvation (0.5% FBS for 2-4 days) followed by 10 hours recovery for G1 phase synchronization. Transgenic cloned embryos were produced through GFPexpressing cell selection and SCNT. The results were the percentage of blastocyst development following SCNT was lower using TBF than BFF cells (23.2% VS 35.2%, P<0.05). No difference in the percentage of cloned blastocysts between the two groups of transgenic nuclear donor of normal and starvation cultures were observed (23.2% VS 18.9%, P>0.05). Two to four GFP-expressing blastocysts were transferred into the uterus of each synchronised recipient. One pregnancy from of seven recipients (21 embryos) was confirmed by rectum palpation 60 days after embryo transfer and one recipient has given birth to a calf at term. PCR and DNA sequencing analysis confirmed that the calf was produced using human proinsulin transgenic animal.
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