A Model Study: Simulation of Marker Gene Deletion and Target Gene Replacement Mediated by Cre/lox System in Cell Line
- VernacularTitle:一种利用Cre/loxP系统进行标记基因删除与靶基因置换的细胞模型研究
- Author:
Wentao CUI
;
Liming REN
;
Jian HOU
;
Ying ZHANG
;
Yongfu CHEN
;
Xiaorong AN
- Publication Type:Journal Article
- Keywords:
lox2272;
loxP;
Cre;
marker gene deletion;
target gene replacement
- From:
Progress in Biochemistry and Biophysics
2008;35(6):650-660
- CountryChina
- Language:Chinese
-
Abstract:
The incorporation of site-specific recombination systems can help to overcome bottlenecks in livestock transgenic technology. For evaluating the efficiency of Cre/lox mediated DNA recombination in embryos and somatic cells, a working model was established using rat mammary carcinoma cell line SHZ-88, aimed at creation of and use repeatedly of selected "friendly loci" in transgenic livestock. An integration vector pTE-lox2272-DsRed-loxP-GFP-loxP, which red fluorescence gene DsRed served as the first target gene and green fluorescence gene GFP as marker gene, was constructed for introduction of acceptor loci in genome. At the same time a replacement vector pT-lox2272-neo-loxP in which Neo coding sequence served as the second target gene was also constructed for replacing DsRed gene. Transgenic cell clones were produced by electroporating SHZ-88 cell with the integration vector. Cells from three transgenic clones selected randomly were further amplified and were then co-electroporated with the replacement vector as well as cre gene. Analysis of the expression patterns of DsRed and GFP indicated that among the 1 070 cell colonies the efficiency on marker GFP deletion was 91.1% and the efficiency on gene replacement was 29.3%. Molecular analysis by PCR and Southern blotting confirmed that the color patterns as expressed by cell colonies could represent the actual molecular events. This working model mediated by Cre/lox system should be useful for the improvement of the present animal transgenic technology.
