TdTomato Flourescent Marker for Visualization in Live Animal Imaging

Sertoli Cells (SC) are immune-privileged, somatic, nurse cells found in the seminiferous tubules of the testes. Besides playing a crucial role in spermatogenesis, they are able to survive when transplanted across immunological barriers; thus establishing their promise as a vehicle for gene therapy. Previously, SC transduced with a recombinant adenoviral vector carrying furin-modified human insulin cDNA were able to restore normal blood glucose levels when transplanted into diabetic mice.  However, insulin expression was transient due to the epichromosomal nature of the adenovirus and normoglycemia was short lived. To accomplish stable insulin expression by incorporating genetic material into the chromosome, a lentiviral vector was created carrying the same insulin (LV-hINS).  After transplantation of the SC-LV-hINS, insulin protein and mRNA were produced at stable levels, however not at high enough levels to restore normoglycemia. Studies are currently on going to increase insulin expression. The lentiviral vector currently being used contains a GFP Fluorescent marker. New imaging technology allows for fluorescent markers to be visualized in live animals, however, GFP is not easily detected. A synthetic tandem double tomato florescent marker has been shown to be extremely bright in live imaging with depths of 1 cm. The goal of this project is to replace the GFP marker with the tdTomato marker through cloning and subcloning, and to create an insulin expression vector.  This expression vector will then be used to create a new lentiviral vector to transduce SC which will then be transplanted in mice that will be imaged live.