学术
基于BAC重组酶系统构建菜航鸡多位点基因打靶载体的研究
唐冬生[1,2] 李芳[1] 蒋泓[1] 胡大林[1] 张细权[3] 李月琴[2] 周天鸿[2]
[1]佛山大学,佛山528000 [2]暨南大学生命科学与技术学院,广州510632 [3]华南农业大学动物科学学院,广州510642
摘 要:
以莱航鸡重复的rDNA基因间的间隔序列为靶位点，利用BAC重组酶系统构建含人干扰素基因的多位点基因打靶载体，为建立莱航鸡多位点基因打靶技术获得关键材料。首先构建BAC-TDN筛选载体，然后构建pYLVS-GID表达载体。将BAC—TDN筛选我体和pYLVS-GID表达我体共转化至大肠杆菌NS3529中，通过其Cre重组酶的作用形成BAC-TDN-VS-GID质粒，采用归位内切酶I-SeeⅠ切除pYLVS质粒骨架，利用接头塔使之环化，构建成莱航鸡大容量多位点基因打靶载体BAC-TDN—GID。每次克隆均经酶切或PCR、测序等鉴定DNA片段的插入及插入方向。以该载体为材料的多位点基因打靶技术将提高基因定点整合效率，解决外源基因不能稳定表达、安全性等部分问题，突破了DNA重复序列不能作为外源基因整合靶位点的禁区。[著者文摘]
文章出处:
《生物工程学报》-2007年23卷2期 -241-245页
栏目信息:
分 类 号:
文献标识码:
A
文章编号:
1000-3061（2007）02-0241-05
Construction of Vector of Multiple Loci Gene Targeting in Leghorn chicken Based on BAC with Crellox P System
TANG Dong-Sheng , LI Fang, JIANG Hong, HU Da-Lin, ZHANG Xi-Quan, LI Yue-Qin, ZHOU Tian-Hong（1 Foshan University, Foshan 528000, China; 2 College of Life Science and Technology, Jinan University, Guangzhou 510632, China; 3 College of Animal Science, Southern China Agriculture University, Guangzhou, China）
Abstract:
Based on the sequence of BAC （Bacterial Artificial Chromosome） along with the Cre/lox P system, the genetargeting vectors to multiple loci of the repetitive internal transcribed spacers between rDNA genes in Leghorn chicken were constructed.The key material of multiple loci gene targeting in vivo would be obtained. First, the plasmid of pYLSV-TDN with TK, HRDS2, and Neo genes was constructed. The TK-HRDS2-Neo DNA fragment obtained from the plasmid of pYLSV-TDN was digested by Not Ⅰ/Hind Ⅲ and inserted into the upstream of the lox P site of BAC plasmid for obtaining the selective vector of BAC-TDN. The expression vector of pYLVS-GID with EGFP, hIFN genes, and HRDS1 was then obtained. The plasmid of BAC-TDN-VS-GID was obtained by cotransformation of the selective vector of BACTDN and the expression vector of pYLVS-GID to E. coli NS3529 through the action of Cre/lox P system. The gene-targeting vector of BAC-TDN-GID to multiple loci of the ITS region in Leghorn chicken was obtained by cleaving the sequence of pYLVS with the homing endonuclease of I -Sce Ⅰ and ligating with the linker of LS. The insertion and the insert direction of DNA fragments were identified by restriction digestion or PCR and sequencing in each clone. The significance of the technique ofgene-targeting vector to multiple loci are shown as follows. First, the targeting loci were increased to 100 - 300. Second, the problems of unstable expression of inserted genes were partially solved. Third, the need for safety against toxicity integration was resolved. Fourth, the forbidden zone of gene integrating on the repetitive DNA sequences was broken through.[著者文摘]
Key words:
gene targeting, multiple loci, vector, BAC, Cre/lox P system, Leghorn chicken, repetitive sequence
基金资助:
国家自然科学基金资助项目（No.30470978,30671194）和广东省自然科学基金资助项目（No.04011645）.
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