一种包含转座子Sleeping Beauty和PiggyBac的新型多功能载体的构建  

Construction of a Novel Multi-functional Vector using Sleeping Beauty and PiggyBac Transposons

作　　者：马元武 廉虹 王冬梅 王贵利 张连峰 

机构地区：[1]中国医学科学院和北京协和医学院,中国医学科学院医学实验动物研究所,卫生部人类疾病比较医学重点实验室 [2]中国医学科学院和北京协和医学院,中国医学科学院医学实验动物研究所,国家中医药管理局人类疾病动物模型三级实验室,北京100021

出　　处：《中国生物化学与分子生物学报》2012年第2期182-189,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：协和青年基金;基本科研业务费(No.DWS201011);卫生部行业基金;实验动物和人类疾病动物模型资源扩展(No.200802036)资助项目~~

摘　　要：DNA转座子作为一种遗传学工具对脊椎动物的转基因、突变体产生、癌基因发现和基因治疗方面都有巨大的贡献.目前,哺乳动物中应用最为广泛、活性最高的DNA转座子为重构于鲑鱼的Sleeping Beauty(SB)转座子和来源于甘蓝蠖度尺蛾(cabbage looper moth Trichoplusia ni)的PiggyBac(PB)转座子.本研究中,我们成功构建了包含PB和SB两种转座子的杂合转座载体,命名为PBSBD.在杂合转座载体中融入了基因捕获框及loxp/Frt元件,用以实现转座过程中的基因捕获和条件性敲除.在HepG2细胞中通过检测报告基因的表达情况及阳性克隆的定位,对构建的杂合转座载体PBSBD进行了活性的初步验证.结果表明,PBSBD能够有效被2种转座酶识别,并能检测到报告基因的表达.本研究所构建的杂合转座载体PBSBD结合2种转座酶,可以应用于大规模筛选突变基因和研究基因功能.并且该杂合转座载体还可以利用SB转座酶的邻近转座特性,结合载体内所包含的loxp/Frt元件用以邻近区域DNA片段的条件性敲除,研究大片段DNA在生物体中的作用.As a useful tool for genetic engineering,DNA transposons have been dedicated totransgenosis,generating mutation,cancer gene discovery and gene therapy.The Sleeping Beauty（SB）reconstructed from salmonoid genome and PiggyBac（PB） modified from the cabbage looper mothTrichoplusia ni have been widely used in mammalian cells for their higher activity than those of thetransposons.The PB transposon and SB transposon were combined to construct a PB /SB hybridtransposon vector named PBSBD containing gene trap cassette and loxp /Frt elements.The activity ofPBSBD was tested in HepG2 cells by monitoring the expression of the report gene and the integration sitein the genome of positive clone.Results showed that PBSBD were effectively identified by twotransposases,and the expression of reporter gene was detected.In the presence of PB transposase and SBtransposase,the PBSBD hybrid transposon vector can be used for many applications,such as large scalegene insertion mutation and gene function research,nested deletion formation using loxp /Frt elementsafter local transposition with SB transposase for tissue specific gene knockout.

关 键 词：转座子 SLEEPING BEAUTY PIGGYBAC 基因工程载体 

分 类 号：Q75[生物学—分子生物学]