High Efficiency Lentiviral Gene Targeting

Abstract

<p>Lentiviral delivery is a highly efficient means of gene transfer. It is not known, however, if simultaneous infection of more than one virus will still be efficient. We hypothesize that simultaneous infection of two lentiviral constructs will infect fertilized single cell zygotes at a high rate of efficiency. To examine this hypothesis, we will determine the rate of infection with a Lv-GFP construct, a Lv-RFP co construct, and following simultaneous infection with Lv-GFP and Lv-RFP constructs. Efficiency of infection will be determined by zygote positivity under epifluorescent microscopy. LvGFP is available, however pLv-RFP must be cloned. To create pLv-RFP, Lv-GFP-VS and the plasmid Pc3-RFP will be double digested using the restriction enzymes BamHI and Apal. The back bone of the GFP plasmid and the RFP insert will then be excised, gel purified, and ligated together. The ligation will then be digested with Spel to disrupt any pLv-GFP that may be present. Following bacterial transformation, colonies will be selected, grown, purified, and analyzed for the correct insert. Lv-RFP will then be used to transfect 293FT cells to produce lentiviral-RFP and virus will be collected. Lentiviral RFP will then be used to determine infection efficiency in Cos7 cells and then in single celled zygotes. This project is of importance in that it will determine the efficiency level of dual infection in zygotes that will subsequently be analyzed in vivo. This elevated level of manipulation provides a higher level of gene targeting with the capability of reducing time from start to finish from 1-2 yrs to 1-2 months. The significance of the analysis will be beneficial to numerous investigators in many disciplines for in vivo analysis of specific gene targeting events.</p><p>This presentation occurred at the Wright State University Campus-Wide Celebration of Research, Scholarship and Creative Activities on April 16, 2010</p>