Quantitation ofin VivoGene Delivery by Restriction Enzyme PCR Generated Polymorphism

Abstract

The Multiple intestinal neoplasia (Min) mouse develops multiple polyps in the intestine, due to a heterozygous mutation of the Apc locus. Our laboratory has been introducing normal human adenomatous polyposis coli (APC) gene into the Min mouse through liposome enema to prevent or reverse polyp formation. We have quantitated the amount of normal human APC gene deliveredin vivoby a restriction enzyme site specific quantitative PCR. Adult Min and BALB/C mice were treated with lipofectant and human APC complementary DNA (cDNA) plasmid. Min colonic DNA was amplified using primers for Apc nucleotide 2524F (5′2524-TCTCGTTCTGAGAAAGACAGAAGCT) and 2679R (5″2679-TGATACTTCTTCCAAAGCTTTGGCTAT). Highlighted primer sequences were purposely different so as to generate twoHindIII restriction enzyme sites in the presence of normal mouse Apc (Apc+). Genomic DNA from untreated Min colonic epithelium revealed two bands: 144 bp for ApcMinand 123 bp for Apc+. BALB/C DNA was amplified using primers flanking a region within the APC gene containing aHindIII site on the human APC, which is absent in the murine APC (Apc). Min's DNA extracted 24 hr after treatment demonstrated a plasmid content of 3% due to a relative increase in the Apc+(123 bp) band. Six weeks of treatments increased delivery to 10%. APC gene therapy of colonic epithelium can be quantitatively measured through restriction enzyme quantitative PCR. Long-term treatment further increases gene delivery. PCR generated polymorphism is a reliable and reproducible technique to initially optimize transfection conditions and ultimately quantitate efficacy in anin vivogene delivery model.