QUANTITATION OF IN-VIVO GENE DELIVERY BY RESTRICTION ENZYME PCR GENERATED POLYMORPHISM

The Multiple intestinal neoplasia (Min) mouse develops multiple polyps in the intestine, due to a heterozygous mutation of the Ape locus. Ou r laboratory has been introducing normal human adenomatous polyposis c oli (APC) gene into the Mill mouse through liposome enema to prevent o r reverse polyp formation. We have quantitated the amount of normal hu man APC gene delivered in vivo by a restriction enzyme site specific q uantitative PCR. Adult Min and BALB/C mice were treated with lipofecta nt and human APC: complementary DNA (cDNA) plasmid. Min colonic DNA wa s amplified using primers for Ape nucleotide 2524F (5('2524)-TCTCGTTCT GAGAAAGACAGAAGCT) and 2679R (5 ''(2679)-TGATACTTCTTCCAAAGCTTTGGCTAT) H ighlighted primer sequences were purposely different so as to generate two HindIII restriction enzyme sites ill the presence of normal mouse Ape (Apc(+)). Genomic DNA from untreated Min colonic epithelium revea led two bands: 144 bp for Apc(Min) and 123 bp far Apc(+). BALB/C DNA w as amplified using primers flanking a region within the APC gene conta ining a HindIII site on the human APC, which is absent in the murine A PC (Apc). Min's DNA extracted 24 hr after treatment demonstrated a pla smid content of 3% due to a relative increase in the Apc(+) (123 bp) b and. Six weeks of treatments increased delivery to 10%. APC gene thera py of colonic epithelium can be quantitatively measured through restri ction enzyme quantitative PCR. Longterm treatment further increases ge ne delivery. PCR generated polymorphism Is a reliable and reproducible technique to initially optimize transfection conditions and ultimatel y quantitate efficacy in an in vivo gene delivery model. (C) 1997 Acad emic Press.