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首页> 外文OA文献 >Combination of the somatic cell nuclear transfer method and RNAi technology for the production of a prion gene-knockdown calf using plasmid vectors harboring the U6 or tRNA promoter
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Combination of the somatic cell nuclear transfer method and RNAi technology for the production of a prion gene-knockdown calf using plasmid vectors harboring the U6 or tRNA promoter

机译：使用携带U6或tRNA启动子的质粒载体将体细胞核转移方法与RNAi技术相结合以生产a病毒基因敲除小牛

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By combining RNAi technology with SCNT method, we attempted to produce transgenic calves with knocked down bPRNP for technological assessments. The respective utilities of type II (tRNA) and type III (hU6) Pol III promoters in mediating plasmid vector-based RNAi for the production of a bPRNP-knockdown calf were compared. Plasmid harboring DNA for siRNA expression was introduced stably into the genome of primary cultured bovine cells. By inserting the transgenic cell into an enucleated bovine egg, SCNT embryos were produced. The ability for SCNT embryos to develop to blastocysts was higher in hU6 based vector groups (44–53%) than in a tRNA group (32%). In all, 30 hU6-embryos and 12 tRNA-embryos were transferred to 11 recipients. Only tRNA-embryos were able to impregnate recipients (six out of 11 transfers), resulting in four aborted fetuses, one stillbirth and one live-born calf. The expression of EGFP, a marker, was detected in all six. The bPRNP transcript levels in the nervous tissues (brain, cerebellum, spinal bulb and spinal cord) from the calf, which was killed 20 days after birth, were reduced to 35% of those of the control calf on average, as determined by qRT-PCR. The PrPC levels, as estimated by western blot were reduced to 86% on average in the nervous tissues. These findings suggest that SCNT technology remains immature, that the tRNA promoter is useful and that RNAi can significantly reduce PRNP mRNA levels, but insufficient reduction of PrPC levels exists in cattle under these conditions.
机译：通过将RNAi技术与SCNT方法相结合，我们尝试生产已敲除bPRNP的转基因小牛，以进行技术评估。比较了II型（tRNA）和III型（hU6）Pol III启动子在介导基于质粒载体的RNAi产生bPRNP基因敲除小牛中的各自效用。将具有用于siRNA表达的DNA的质粒稳定地引入原代培养的牛细胞基因组中。通过将转基因细胞插入去核的牛卵中，产生了SCNT胚胎。基于hU6的载体组（44–53％）的SCNT胚胎发育为胚泡的能力高于tRNA组（32％）。总共将30个hU6-胚胎和12个tRNA胚胎转移到11个受体中。只有tRNA胚胎能够浸渍受体（11次转移中有6次），从而导致4胎胎儿流产，1例死胎和1胎活牛犊。在全部六个中检测到标志物EGFP的表达。小牛的神经组织（大脑，小脑，脊髓和脊髓）中的bPRNP转录水平在出生后20天被杀死，平均降低至对照小牛的35％（根据qRT- PCR。 Western blot估计，神经组织中的PrPC水平平均降低到86％。这些发现表明，SCNT技术仍然不成熟，tRNA启动子是有用的，RNAi可以显着降低PRNP mRNA的水平，但是在这些条件下牛体内PrPC水平的降低不足。

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