LE3 .A278 2009

2009

Wilson, Brian

Acadia University

Bachelor of Science

Honours

Biology

Biology

Cre recombinase is an enzyme that recognizes loxP (Locus of Crossover in P1; 34 base pairs length) sites inserted into a target gene that flank a specific sequence of DNA. This enzyme first cuts a DNA sequence that is encompassed by flanking loxP (floxed) sites and then rejoins the two ends of DNA resulting in recombination of a gene. When the Cre recombinase is fused with the Tat protein (11 amino acids), the resultant fusion protein penetrates cells and recombines a floxed gene. This approach has been used to inactivate floxed genes in order to determine their function within various types of cells. The goal of the present study was to induce E. coli cultures to produce the Tat-Cre fusion protein, obtain purified fractions and use them to recombine genes that had been floxed in murine astrocytic cultures. Tat-Cre protein production was induced in E. coli strain BL21(DE3)pLysS and purified by immobilized metal ion affinity chromatography and ion exchange chromatography. Purity of Tat-Cre isolated in this manner was confirmed by SDS-PAGE. The activity of Tat-Cre was then assayed by incubation with murine astrocytes that had been genetically engineered such that the hypoxia-inducible factor gene HIF-2α had been floxed. Tat-Cre successfully produced recombination of these genes at concentrations as low as 200nM. Tat-Cre will be used in subsequent experiments to determine whether functional loss of the transcriptional regulatory factors HIF-1 and/or HIF-2 impairs the ability of astrocytes and neurons to produce proteins such as erythropoietin and vascular endothelial growth factor that enable these cells to survive in a low oxygen environment.

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