Plcd1转基因小鼠的制备  

Establishment of Plcd1 Transgenic Mice

作　　者：杨伟伟[1] 殷筱舒 殷黎静[3] 张艳丽[1] 李高天[1] 刘桂杰[1] 俞利平[1] 顾美儿[1] 吴宝金[1] 

机构地区：[1]杭州师范大学实验动物中心,浙江杭州310036 [2]山东省青岛第十七中学,山东青岛266031 [3]江苏大学实验动物中心,江苏镇江212013

出　　处：《杭州师范大学学报（自然科学版）》2013年第1期1-7,共7页Journal of Hangzhou Normal University(Natural Science Edition)

基　　金：国家自然科学基金项目(31071092);浙江省公益性技术应用研究计划项目(2012C37084);浙江省大学生科技创新活动计划(新苗人才计划)2011R421029

摘　　要：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.In order to generate Plcd1 transgenic mice for the functional study of Plcd1gene, the 2.2kb total length of Plcd1 gene was RT-PCR amplified and cloned into T vector to construct pMD19-T-Plcd1vector at first. The target Plcd1 fragment with proper restriction endonuclease sites was obtained by PCR amplification using specially designed primers and pMD19-T-Plcd1 template. This target Plcd1 fragment and pEF6/VS-His vector were digested respectively by Speland BstBI in the same time, and then were connected together to construct pEF6/V5-His-Plcd1 vector. After it was verified to express in L929 cells, 4.5Kb length of transgenic construction including hEF-1α promoter,Plcd1, V5, His-tag and BGH was isolated and microinjected into 681 zygotes to produce 82 offspring. Finally, 13 hereditable lines were established from 15 positive transgenic founder mice. Western Blotting experiment revealed that PLCD1-HIS fusion protein was expressed in testicular tissue but not in the skin. The Plcd1 transgenie mice provide potential material for the functional study of Plcd1.

关 键 词：Plcd1 pEF6 V5-His-Plcd1 显微注射 转基因小鼠 

分 类 号：Q95-33[生物学—动物学]