Liu X D, Sun Z Q, Zhang C B, Wu H L
College of Biology, Peking University, Beijing 100871, China.
Yi Chuan Xue Bao. 2001;28(6):493-501.
In order to get CgA gene antisense DNA transgenic mouse, we constructed the CgA gene antisense DNA plasmid pCAS2C and microinjected it into the female pronucleus of fertilized mouse eggs, and transplanted them into oviduct of the foster. Every offspring of the fosters was determined by the PCR method. The positive mice had a 300 bp DNA electrophoresis band. We selected two male positive mice from 50 offspring survived of the pseudomother. Then, two positive mice crossed with normal mice respectively to reproduce offspring of F1. All offspring of F1 were determined by PCR to select positive offspring. Positive offspring of F1 carried only one allele of pCAS2C (heterozygous pCAS2C/-). Positive F1 offspring were selfcrossed, 1/4 offspring of F2 carrying two unites of one allele pCAS2C (pCAS2C/pCAS2C) are homozygous. Then, all offspring of homozygous F2 crossed with normal mice, could produce 300 bp DNA electrophoresis band by PCR. Total RNA of brain tissue of transgenic mouse was used to RT-PCR method, the 300 bp DNA product was obtained. The result indicates that the reading frame of CgA antisense DNA of pCAS2C has expressed in the transgenic mice.
为获得CgA基因反义DNA转基因小鼠，我们构建了CgA基因反义DNA质粒pCAS2C，并将其显微注射到小鼠受精卵的雌原核中，然后将其移植到代孕母鼠的输卵管中。通过PCR方法对代孕母鼠的每只后代进行检测。阳性小鼠有一条300 bp的DNA电泳条带。我们从50只代孕母鼠存活的后代中挑选出两只雄性阳性小鼠。然后，将两只阳性小鼠分别与正常小鼠杂交以繁殖F1代后代。通过PCR对F1代的所有后代进行检测以挑选出阳性后代。F1代的阳性后代仅携带一个pCAS2C等位基因（杂合子pCAS2C/-）。F1代阳性后代进行自交，F2代中有1/4携带两个pCAS2C等位基因单位（pCAS2C/pCAS2C）的后代为纯合子。然后，所有纯合子F2代后代与正常小鼠杂交，通过PCR可产生300 bp的DNA电泳条带。用转基因小鼠脑组织的总RNA进行RT-PCR方法，获得了300 bp的DNA产物。结果表明pCAS2C的CgA反义DNA的阅读框在转基因小鼠中得到了表达。
