Transgene excision in zebrafish using the phiC31 integrase.
Genome engineering strategies employing site-specific recombinases (SSRs) have become invaluable to the study of gene function in model organisms. One such SSR, the integrase encoded by the Streptomyces bacteriophage phiC31, promotes recombination between heterotypic attP and attB sites. In the present study I have examined the feasibility of the use of phiC31 integrase for intramolecular recombination strategies in zebrafish embryos. I report here that (1) phiC31 integrase is functional in zebrafish cells, (2) phiC31 integrase can excise a transgene cassette flanked by an attB and an attP site, analogous to a common use of the Cre/lox SSR system, (3) phiC31 integrase functions in the zebrafish germline, and (4) a phiC31 integrase-estrogen receptor hormone-binding domain variant fusion protein catalyzes attB-attP recombination in zebrafish embryos in a 4-hydroxytamoxifen-dependent manner, albeit less efficiently than phiC31 alone. These features should make this a useful approach for genome manipulations in the zebrafish.
Lister
James A
JA
Department of Human and Molecular Genetics, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298-0033, USA. jalister@vcu.edu
D000818:Animals; D030801:Animals, Genetically Modified; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian; D005240:Feasibility Studies; D005456:Fluorescent Dyes / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D008845:Microinjections; D016366:Open Reading Frames; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011995:Recombination, Genetic; D019076:Transgenes; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism / Q000821:virology
D005456:Fluorescent Dyes; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D019426:Integrases