Date of Award

Fall 12-1-2004

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Life Sciences

First Advisor

Gary W. Stuart

Second Advisor

Swapan Ghosh

Third Advisor

Kathleen Dannelly

Abstract

Current transgenesis has become easier for certain species due to the recent development of exogenous non site-specific recombination systems such as Sleeping Beauty (SB) transposon, and site-specific recombination systems such as Cre-loxP. Such recombination systems overcame two major problems caused by conventional transgenesis, position effect and copy number effect. SB is a reconstructed fish transposon that has shown to efficiently insert transgenes into heterologous mammalian chromosomes. The Cre-loxP system is frequently used to delete a given sequence from the genome or to target the integration of foreign DNA. The use of both systems in combination, however, has not yet been attempted in any organism. In order to improve the efficiency, flexibility, and reproducibility with which genetically engineered organisms are produced, we are attempting to simultaneously exploit these two wellcharacterized single copy recombination systems in transgenic zebrafish. In this study, function and compatibility of four loxP variants have been analyzed by performing excision and inversion assays in Cre-expressing E.coli. None of the loxP variants tested here showed any compatibility with the others. The loxA site showed relatively high recombination efficiency compared to other loxP variants. In addition to a loxP site, a loxA site was inserted into our transformation vectors to facilitate the Cremediated gene exchange. To test the efficiency of the two-recombination systems in zebrafish, one-cell or two-cell zebrafish embryos were microinjected with transformation vectors containing elements of the Cre-loxP system and/or the SB transposon system. IV Transformation vectors also included a reporter gene cassette (GFP or RFP coding sequence) to facilitate the detection of recombination events. ere-mediated recombination efficiency was estimated at 75% to 100% depending on the concentration of ere mRNA. Microinjection of ere mRNA appeared to be slightly more effective for this purpose than microinjection of ere-expressing plasmid DNA. Although SB-mediated excision and integration were not observed by transient assay, they will likely be detected in the stable transgenic zebrafish generated in this study. These fish should also be useful for observing SB-mediated mobilization of trans genes. Overall, our study provided the first evidence that ere recombinase can mediate effective, site-specific deletion of stable transgenes in zebrafish. It also suggested that efficient and precise genome engineering in zebrafish will be possible in the future by using ere recombinase and SB transposase.

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