Efficient gene activation system on mammalian cell chromosomes using recombinant adenovirus producing Cre recombinase.
To develop a method for activating genes located on cell chromosomes, an on/off switching unit regulated by the site-specific recombinase Cre was constructed. The switching unit was designed to express firstly the neo gene and secondly the reporter lacZ gene by Cre-mediated excisional deletion of the neo gene. CV1 cell lines bearing the switching unit on a cell chromosome were isolated and activation of the lacZ gene was examined after infection with a Cre-producing recombinant adenovirus. In one cell line virtually 100% of the cells stably expressed the lacZ gene, whereas in another cell line lacZ-expressing cell populations reached only to about 90% and decreased after cell divisions. The Southern blot analyses showed that the latter type of cells contained a head-to-tail array of the switching units, and that consequently the lacZ-expressing units were excised from a cell chromosome and present as extrachromosomal circular DNAs. These results showed that the system offers efficient activation of genes introduced into cell chromosomes and that the organization of the reporter units are important for efficiency and duration of the activated gene expression.
Kanegae
Y
Y
Laboratory of Molecular Genetics, University of Tokyo, Japan.
Takamori
K
K
Sato
Y
Y
Lee
G
G
Nakai
M
M
Saito
I
I
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D002875:Chromosomes; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D007763:Lac Operon; D008322:Mammals; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D015533:Transcriptional Activation; D019076:Transgenes; D014764:Viral Proteins
D011994:Recombinant Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases