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通过制备Plcd1转基因小鼠研究Plcd1基因功能.首先逆转录PCR获得约2.2kb的Plcd1基因全长,T载体克隆后测序验证;以pMD19-T-Plcd1为模板,通过设计引物及PCR扩增引入酶切位点,与pEF6/V5-His同时进行酶切、连接,构建pEF6/V5-His-Plcd1表达载体;经真核表达验证后,酶切获得目标片段,显微注射681枚受精卵,在82只仔鼠中获得转基因阳性首建鼠15只,其中13只稳定遗传并建系.PLCD1-HIS融合蛋白在睾丸组织中表达,在皮肤组织中无表达.Plcd1转基因小鼠为Plcd1功能研究奠定了基础.
The Plcd1 gene function was studied by preparing Plcd1 transgenic mice.After that, the full-length Plcd1 gene of about 2.2kb was obtained by reverse transcription PCR, and the T vector was cloned and verified by sequencing. Using pMD19-T-Plcd1 as a template, The pEF6 / V5-His-Plcd1 expression vector was digested and ligated with pEF6 / V5-His at the same time. After the eukaryotic expression verified, the target fragment was digested with 681 zygotes Of the 82 offspring mice, 15 transgenic mice were obtained, of which 13 were stable and inherited and established.PLCD1-HIS fusion protein was expressed in the testis tissue but not in the skin tissue.Plcd1 transgenic mice lay the foundation for the function of Plcd1 The foundation.