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背景:近年的研究表明,在脑缺血再灌注损伤机制中,bcl-xl基因可能起着抗神经细胞凋亡的脑保护作用。目的:观察转bcl-xl基因小鼠外源基因的复制及传代稳定性问题。设计:以基因为观察对象。单位:华中科技大学同济医学院附属同济医院神经科和湖北省农科院畜牧兽医研究所。材料:实验于1999-10/2001-04在华中科技大学同济医学院附属同济医院完成。选取昆明种小鼠4只。方法:通过显微注射法建立转人bcl-xl基因小鼠,将PCR及Southern-blot检测整合阳性的小鼠与正常鼠交配并传代,然后检测子代小鼠外源基因的表达情况。作Southern-blot检测,结果证实第10,13,5,6号小鼠为整合阳性小鼠。以4只整合阳性小鼠作为首建者,分别与正常的昆明白小鼠交配繁殖传代,所生子代记为F1代,各系列的F1～F4代均采用阳性转基因小鼠与正常的昆明白小鼠交配繁殖传代方法,保持其杂合子形式。主要观察指标:检测外源基因的复制和传代的稳定性。结果:10号小鼠传代过程中外源基因PCR检测阳性率保持稳定,符合孟德尔遗传规律,表明外源基因能够稳定遗传。6号小鼠PCR阳性率呈轻微下降趋势,但仍能够保持相对稳定遗传。13号和5号小鼠PCR阳性率均呈明显的下降趋势,表明这2只小鼠存在外源基因遗传丢失现象。结论:应用基因组DNA做目的基因,可获得能够稳定遗传的转基因鼠系。
BACKGROUND: Recent studies have shown that the bcl-xl gene may play a neuroprotective role against neuronal apoptosis in the mechanism of cerebral ischemia-reperfusion injury. OBJECTIVE: To observe the replication and passage stability of exogenous gene of bcl-xl transgenic mice. Design: Take gene as object of observation. Unit: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology and Institute of Animal Husbandry and Veterinary Medicine, Hubei Academy of Agricultural Sciences. MATERIALS: Experiments were performed at Tongji Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology from October 1999 to April 2001. Select Kunming mice 4. Methods: The bcl-xl transgenic mice were established by microinjection method. The positive and negative mice were passaged and passaged by PCR and Southern-blot. The expression of foreign genes was detected by PCR. Southern blot results showed that mice No. 10, No. 13, No. 5 and No. 6 were positive for integration. Four integrated positive mice were used as the founder, respectively, and the normal Kunming white mice were passaged and passaged. The offspring were recorded as F1 generation, F1 ~ F4 of each series were positive transgenic mice and normal Kunming white Mating mice are passaged and maintained in their heterozygous form. MAIN OUTCOME MEASURES: Detection of foreign gene replication and passage stability. Results: The positive rate of exogenous gene PCR in 10 # mice kept stable, which accorded with Mendel ’s genetic rule, indicating that the foreign gene could be stably inherited. PCR positive rate of 6 mice showed a slight downward trend, but still able to maintain a relatively stable genetic. PCR positive rates of mice No. 13 and No. 5 showed a significant downward trend, indicating that there was a genetic loss of foreign genes in the two mice. Conclusion: Genomic DNA can be used as the target gene to obtain a stable transgenic mouse line.