Transfection of Primary Kidney Cells with Mutated SV40 T ag DNA Containing a CCKCR Intron-Derived Sequence
Date of Award
Cholecystokinin-C receptor (CCKCR) is an alternatively spliced version of cholecystokinin-B receptor (CCKBR) found in pancreatic cells. CCKCR is uniquely expressed by cancer cells, making it an important target for immune therapy. Simian Virus 40 Large Tumor Antigen (SV40 T ag) has proven to be a powerful model in the study of cell-mediated immune responses to tumors. Our goal was to construct cells which express a derivative of SV40 T ag that incorporates the retained intron sequence from CCKCR to use as a tool in the study of cellmediated immunity targeting this unique sequence. Two variations of plasmids containing mutated T ag DNA with a 20 codon replacement (20mer) from CCKCR intron 4 were prepared: containing either an F408A substitution in epitope IV (pCBKG/F408A) or wild-type (WT) epitope IV (pCBKG/WT). Sequencing confirmed the presence of the 20mer in place of epitope I through II/III, confirmed the appropriate sequence in epitope IV (F408A or WT), and revealed a previously undetected silent mutation within the 20mer. These DNA preparations were purified and used to transfect primary kidney cells harvested from naïve mice. Transfection was performed in four parallel batches: pCBKG/F408A, pCBKG/WT, pSelectESV (WT Tag), and a no DNA control. After several weeks, dense foci of growth were isolated and expanded. Those transfected with WT T ag showed greater numbers of foci and growth rate of isolated cell lines than those transfected with either 20mer replacement mutant. Immunofluoresence revealed successful transfection of the WT, but was inconclusive for pCBKG/WT and negative for pCBKG/F408A.
Grando, Kaitlyn; Burkett, Courtnry; and Mylin, Lawrence, "Transfection of Primary Kidney Cells with Mutated SV40 T ag DNA Containing a CCKCR Intron-Derived Sequence" (2015). Honors Projects and Presentations: Undergraduate. 169.