title: Transgene manipulation in zebrafish by using recombinases.
abstract: Although much remains to be done, our results to date suggest that efficient and precise genome engineering in zebrafish will be possible in the future by using Cre recombinase and SB transposase in combination with their respective target sites. In this study, we provide the first evidence that Cre recombinase can mediate effective site-specific deletion of transgenes in zebrafish. We found that the efficiency of target site utilization could approach 100%, independent of whether the target site was provided transiently by injection or stably within an integrated transgene. Microinjection of Cre mRNA appeared to be slightly more effective for this purpose than microinjection of Cre-expressing plasmid DNA. Our work has not yet progressed to the point where SB-mediated mobilization of our transgene constructs would be observed. However, a recent report has demonstrated that SB can enhance transgenesis rates sixfold over conventional methods by efficiently mediating multiple single-copy insertion of transgenes into the zebrafish genome (Davidson et al., 2003). Therefore, it seems likely that a combined system should eventually allow both SB-mediated transgene mobilization and Cre-mediated transgene modification. Our goal is to validate methods for the precise reengineering of the zebrafish genome by using a combination of Cre-loxP and SB transposon systems. These methods can be used to delete, replace, or mobilize large pieces of DNA or to modify the genome only when and where required by the investigator. For example, it should be possible to deliver particular RNAi genes to well-expressed chromosomal loci and then exchange them easily with alternative RNAi genes for the specific suppression of alternative targets. As a nonviral vector for gene therapy, the transposon component allows for the possibility of highly efficient integration, whereas the Cre-loxP component can target the integration and/or exchange of foreign DNA into specific sites within the genome. The specificity and efficiency of this system also make it ideal for applications in which precise genome modifications are required (e.g., stock improvement). Future work should establish whether alternative recombination systems (e.g., phiC31 integrase) can improve the utility of this system. After the fish system is fully established, it would be interesting to explore its application to genome engineering in other organisms.
authors.affiliation: Department of Life Sciences, Indiana State University, Terre Haute, Indiana 47809, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Dong
authors.forename: Gary W
authors.initials: GW
authors.lastname: Stuart
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000737:chemistry; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional / Q000379:methods*; D045522:Recombinases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D019076:Transgenes / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics*
chemical_list: D045522:Recombinases; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases