缺失糖基磷脂酰肌醇锚的Doppel转基因小鼠的构建

Foundation of Transgenic Mice Expressing Doppel without Glycosyl Phosphatidyl Inositol

将缺失糖基磷脂酰肌醇(glycosyl phosphatidyl inositol,GPI)的Doppel的质粒(pDoppel1-155),用NotⅠ酶切,回收并纯化目的基因片段,通过显微注射,导入BALB/c小鼠受精卵的雄原核内,结果32只仔鼠出生,其中有6只PCR检测阳性。将其中1只F0代小鼠与正常小鼠交配,共获得21只F1代小鼠,经PCR检测,有10只仔鼠为阳性。对2只F1代PCR阳性小鼠脑组织进行RT-PCR检测,其中1只为阳性。采集3只F1代PCR阳性小鼠脑组织进行Westernblotting检测,在小鼠脑组织能够表达17ku的Doppel1-155蛋白。本研究为进一步深入研究Doppel蛋白在体内的生物学特性提供了转基因动物模型。

A recombinant Doppel plasmid without glycosyl phosphatidyl inositol （pDoppel 1-155） was digested by Not I ,then the objective gene was recovered and purified. The gene was introduced into male pronucleus of BALB/c mouse fertilized ovum by microinjection, then we got 32 mice which have been injected the Dpl 1-155 gene, in which 6 was positive by PCR ; one F0 generation of Tg mouse expressing Dpl without GPI was mated with a wild-type mouse, then 21 F1 generation mouse were got, in which 10 were positive by PCR identification. Brain tissue of three F1 generation mice which were positive by PCR identification was collected, and was identified by Western blotting, there was 17 ku Dpl 1-155 protein expressed in the brain. So we have generated Tg mice expressing Dp1 without GPI which provides Tg animal models for investigating the bionomics of Dpl protein.