两种含SV40T不同区段的基因构建及其转基因小鼠的建立

The Construction of Two Gene Containing Different Fragments of SV40T and the Development of Its Transgenic Mice

目的 为解决SV4 0T转基因小鼠高发瘤难保种的问题 ,构建了两种含有SV4 0T不同区段的外源基因 :Rb结合域点突变的SV4 0T基因 (SV4 0T DRb)和无p5 3结合域的SV4 0T基因 (SV4 0T Dp5 3)。方法 利用分子生物学的基因克隆手段 ,将改造的SV4 0T基因片段克隆测序 ,最终将这两个改造后的基因克隆进乳腺特异性的真核表达载体p2 0 5C3中 ,运用雄原核显微注射法制备转基因小鼠。结果 利用PCR方法检测出 6只双阳性转基因 (同时检测到SV4 0T DRb和SV4 0T Dp5 3两种基因 )小鼠 ,为避免检测结果中假阳性的发生 ,应用Southern blot方法检测出 1只双阳性转基因小鼠。结论 本试验的结果证明 ,构建的两种含SV4 0T不同区段的策略是成功的 ,其建立的阳性转基因小鼠确实是弱化了SV4 0T转基因小鼠高发瘤的特性。

Objective To overcome difficulties in passage for SV40T transgenic mice, two genes containing different fragments of SV40T were constru cted: one gene is called SV40T-DRb which was mutated in Rb motif of SV40 T, another gene is called SV40T-Dp53 which was deprived of p53-binding d omain. Methods U sing recombi nant DNA techniques, the two changed gene fragments of SV40T were sequenced and cloned, and finally the two genes were cloned into the mammary gland-specific e x pression vector-p205C3. Microinjection of the expression vectors into male nucl e i was used to establish transgenic mice. PCR and Southern-blot were used to det e ct the positive transgenic mice, avoiding the occurrence of false positive trang enic mice. Results Six transgenic mice that were positive for b oth gene SV40T-DRb and gene SV40T-Dp53 were detected by PCR. One transgenic mouse that was positive for both gene SV40T-DRb and gene SV40T-Dp53 was detected by Southern blot. Co nclusion The above mentioned findings suggested that the two gene const ructs con taining different fragments are successful and the transgenic mice are surely wi th reduced tumorigenesis of SV40T.