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Jesseca Bullock

Fellow – Jesseca Bullock

Mentor – Charles Long

Project – Production of Transgenic Cats Resistant to Feline Immunodeficiency Virus

Objective – Improve the production of cats resistant to Feline Immunodeficiency Virus (FIV) through genetic engineering. Aim 1: to improve the efficiency of in vitro feline oocyte maturation by arresting oocytes prior to maturation. Aim 2: to design and express short hairpin RNAs (shRNAs) targeting FIV genes in feline cells.

Animals or Sample Population – Oocytes from routinely ovariohysterectomized felines were used for Experiment 1. A feline T-cell line (Mya-1) was used for Experiment 2.

Procedure – Two experiments were performed: Experiment 1 was to improve oocyte maturation by arresting immature feline oocytes for 24 hours. The addition of two arresting agents followed by 20, 24, 28, and 32 hours of maturation was compared to the same time points without arrest. Experiment 2 involved designing and producing shRNA targeting highly-conserved portions of the FIV genome. Using self-inactivating lentiviral vectors containing a fluorescent marker, the shRNAs were stably integrated into Mya-1 and HEK293 cells.

Results – In Experiment 1, no statistical difference between arrested and unarrested oocytes was found due to uncontrollable variability. For Experiment 2, Mya-1 cells and HEK293 cells were successfully transduced, as verified by fluorescent microscopy.

Conclusions and Clinical Relevance – More work is needed on both fronts of this project. In future work, experience is an important variable to consider in selection of grade A and B oocytes. Increasing the recombinant virus titer, using a gamma retrovirus, and/or raising the virus in various media are some techniques being considered for improving transduction rates in Mya-1 cells.