CAJ | 학술논문

跨膜蛋白66(TMEM66)是与细胞凋亡以及癌症发生密切相关的重要功能基因,为了在个体水平研究其生物学功能,我们进行了TMEM66基因突变体(TMEM66V)转基因小鼠的构建.本研究通过原核注射法进行转基因操作,将获得的312个受精卵移植到13只代孕母鼠中.运用PCR、Southern blotting对出生的小鼠进行转基因鉴定,对于转基因阳性小鼠通过传代试验研究外源基因是否稳定整合,并通过反向PCR方法研究外源基因的整合方式.结果显示在出生的55只小鼠中有6只为转基因阳性,其中3只转基因小鼠可以稳定传代,表明这些小鼠中外源基因发生了稳定整合.反向PCR检测结果发现外源片段是以串联重复的方式整合到转基因小鼠的基因组中.本研究成功构建了TMEM66基因突变体转基因小鼠,为进一步研究TMEM66基因的功能奠定了基础.
과막단백66(TMEM66)시여세포조망이급암증발생밀절상관적중요공능기인,위료재개체수평연구기생물학공능,아문진행료TMEM66기인돌변체(TMEM66V)전기인소서적구건.본연구통과원핵주사법진행전기인조작,장획득적312개수정란이식도13지대잉모서중.운용PCR、Southern blotting대출생적소서진행전기인감정,대우전기인양성소서통과전대시험연구외원기인시부은정정합,병통과반향PCR방법연구외원기인적정합방식.결과현시재출생적55지소서중유6지위전기인양성,기중3지전기인소서가이은정전대,표명저사소서중외원기인발생료은정정합.반향PCR검측결과발현외원편단시이천련중복적방식정합도전기인소서적기인조중.본연구성공구건료TMEM66기인돌변체전기인소서,위진일보연구TMEM66기인적공능전정료기출.
Transmembrane protein 66 play important roles in cell apoptosis and tumorigenesis. In order to investigate the function of TMEM66 gene, the transgenic mouse model containing the mutant TMEM66 gene was constructed in this study. The transgenic mice were generated by u-sing microinjection method and 312 injected zygotes were implanted into 13 foster mothers. The positive transgenic mice were confirmed by PCR and Southern blotting. Also, the stability of the transgene was confirmed by passage and the way of integration of the transgene was studied using inverse PCR method. In this study, 6 positive individuals among the 55 neonatal pups were found by Southern blotting. Three positive mice were found to be able to transfer the transgene to next generation. This result indicted that the transgene was integrated into host genome stably. In-verse PCR results indicated that the transgenic DNA fragment was integrated into the host ge-nome in a serial multi-copy manner. Therefore, the transgenic mice model containing the mutant TMEM66 gene was constructed successfully. This study offered useful model for further investi-gation of the function of TMEM66 gene.