141 DEVELOPMENT OF rLENTIVIRAL VECTORS FOR MALE GERMLINE-SPECIFIC Cre RECOMBINASE EXPRESSION IN LIVESTOCK

Abstract

Transgenic livestock have been used as biomedical models and have the potential to increase production characteristics. Unfortunately, some of the tools used to confirm genetic modification (transgenesis) are unacceptable in terms of public image. One key component is a fluorescent marker confirming foreign gene insert. The fluorescent protein benefits the researchers producing and selecting transgenic animals but is not required for the enhancement of the animal. Removal of the fluorescent marker can be accomplished by employing the Cre-lox recombination system. By using this system one can produce male genetically modified animals that express the enhanced trait in addition to the fluorescent marker, but their sperm only contain the portion of the transgene that represents the enhanced production trait. As a result, offspring derived from these animals exhibit the desired production trait but not the fluorescent marker. The goal of this research was to develop rlentiviral vectors that use the gamete-specific promoter stimulated by retinoic acid 8 (Stra8) to drive expression of Cre recombinase. The base rlentiviral vector we chose to use was pLB. It is ideal for this research because it has a U6 promoter to drive expression of a short hairpin RNA for an enhanced production trait, as well as Lox P sites flanking a cytomegalovirus-green fluorescent protein (CMV-GFP) expression cassette. Initially we identified and PCR amplified a 400-bp mouse Stra8 promoter and a 1.5-kb promoter region of the pig. The Stra8 promoters were integrated into the pLB vector directly upstream of the green fluorescent protein (GFP). These intermediate vectors should have germline-specific expression of GFP and are the first vectors using a pig Stra8 promoter. Next, we PCR amplified and inserted the coding sequence for Cre recombinase into these vectors for germline-specific Cre expression resulting in the first pig Stra8-Cre expression vector. The Lox P sites of this vector are flanking the GFP expression cassette as well as the Str8-Cre expression cassette. To confirm the functionality of the Cre-lox recombination system, the pLB vectors were transfected into human embryonic kindey 293T cells and fluorescence was measured. After Day 2, a Cre expression plasmid was transfected and 3 days post-Cre transfection, fluorescence was measured again. A decrease in fluorescence and GFP positive cell numbers was observed, thus confirming the functionality of the Cre-lox recombination event for these vectors. These vectors will be used to produce transgenic animals by lentiviral transgenesis. Our laboratory has been successful in using this method to produce transgenic livestock. The transgenic animals will be analysed to confirm male germ-cell-specific expression of Cre as well as removal of the GFP fluorescence. If successful, these will be the first transgenic animals using a pig Stra8 promoter for Cre expression for a novel single rlentiviral vector Cre-lox recombination system.