Vill转基因小鼠的制备

摘要:目的　通过制备Vill转基因小鼠研究该基因的功能。 方法与结果　首先由RT-PRC方法获得全长约2605bp的Vill基因；经T载体克隆测序验证后，以克隆载体pMD19-T-Vill为模板，设计引物并引入酶切位点，将PRC扩增产物与pEF6/V5-His-LacZ同时进行酶切、连接，构建表达载体pEF6/V5His-Vill；经真核表达验证后，酶切获得含Vill基因的显微注射DNA构件；显微注射390枚受精卵后，在出生存活的77只仔鼠中获得转基因阳性GO代小鼠19只，其中16只能够稳定遗传并建系，转基因阳性小鼠外观未有明显改变。 结论　Vill转基因小鼠为该基因的功能研究准备了实验材料。

Establishment of Vill Transgenic Mouse

In order to generate Fill transgenic mouse for the functional study of Vill gene, the 2 605bp total length of Fill gene was amplified by RT-PCR and cloned into the pMD19-T vector to construct pMD19-T-Vill vector at first. The target Fill fragment with proper restriction endonuelease sites was obtained by PCR amplification using specially designed primers and pMD19-T-Vill template. This target Fill fragment and pEF6/VS-His vector were digested respectively by SpeI and BstBI in the same time, and then were connected together to construct pEF6/V5-His-Vill expression vector. After target gene was verified to express in L929 cells, 4.5 kb length of transgenic construction including hEF-1 α promoter,Plcdl ,V5 ,His-tag and BGH was isolated and microinjected into 390 zygotes to produce 77 offspring. Finally, 16 hereditable lines were established from 19 positive transgenic founder mice. General appearance was not found difference between Vill-transgenic mice and the wild type mice. The Fill transgenic mouse is potential material for the study of Vill gene functions.