Site-specific gene integration in cultured silkworm cells mediated by phiC31 integrase.
The integrase from the Streptomyces bacteriophage phiC31 carries out efficient recombination between an attP site in the phage genome and an attB site in the host chromosome. In the present study, we have used the phiC31 integrase system to mediate site-specific recombination in the cultured silkworm cell line BmN4. A plasmid containing a cDNA encoding DsRed flanked by two phiC31 attP sites was co-transfected together with a helper plasmid encoding the phiC31 integrase into a cell line in which phiC31 attB sites inserted between a baculovirus IE2 promoter, and a polyadenylation signal are present in one chromosome. Seven days after transfection, expression of DsRed was observed in transformed cells. Nucleotide sequence analysis demonstrated that the expected recombination between the attB and attP sites had been precisely carried out by the phiC31 integrase. These results indicate that the phiC31 site-specific recombination system should be widely applicable for efficient site-specific gene integration into silkworm chromosomes.
Nakayama
Gaku
G
Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki, 812-8581 Fukuoka, Japan.
Kawaguchi
Yutaka
Y
Koga
Katsumi
K
Kusakabe
Takahiro
T
D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics*; D017103:Bacillus Phages / Q000201:enzymology*; D016367:Baculoviridae / Q000235:genetics; D012831:Bombyx / Q000235:genetics*; D002460:Cell Line; D015870:Gene Expression; D018390:Gene Targeting; D019426:Integrases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D039104:RNA 3' Polyadenylation Signals / Q000235:genetics; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000821:virology; D014764:Viral Proteins / Q000235:genetics*
D014764:Viral Proteins; D019426:Integrases