Related Experiment Videos
Quantitation of in vivo gene 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 delivered in vivo by 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 two HindIII restriction enzyme sites in the presence of normal mouse Apc (Apc+). Genomic DNA from untreated Min colonic epithelium revealed two bands: 144 bp for ApcMin and 123 bp for Apc+. BALB/C DNA was amplified using primers flanking a region within the APC gene containing a HindIII 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 an in vivo gene delivery model.
Insights
Gene therapy using normal human adenomatous polyposis coli (APC) gene delivered via liposome enema successfully increased APC gene delivery in mice with intestinal polyps. Long-term treatment enhanced gene delivery, showing promise for APC gene therapy in colon cancer prevention.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- Multiple intestinal neoplasia (Min) mice develop intestinal polyps due to a mutated Apc gene.
- Adenomatous polyposis coli (APC) gene is crucial for colon health.
- Gene therapy aims to introduce functional APC to counteract polyp formation.
Purpose of the Study:
- To quantitatively measure the delivery of normal human APC gene into Min mice using liposome enema.
- To assess the efficacy of APC gene therapy in preventing or reversing intestinal polyp formation.
- To establish a reliable method for optimizing gene delivery conditions.
Main Methods:
- Utilized restriction enzyme site-specific quantitative PCR to quantify human APC gene delivery in vivo.
- Amplified Min colonic DNA using specific primers designed to create restriction sites.
- Analyzed DNA bands representing ApcMin and Apc+ alleles to determine gene delivery efficiency.
Main Results:
- Initial treatment showed 3% plasmid content, indicated by an increased Apc+ band.
- Six weeks of treatment increased gene delivery to 10%.
- Restriction enzyme quantitative PCR proved effective for measuring APC gene therapy in colonic epithelium.
Conclusions:
- APC gene therapy can be quantitatively measured in colonic epithelium.
- Long-term treatment with liposome-delivered APC gene enhances gene delivery efficacy.
- PCR-generated polymorphism is a reliable technique for optimizing and quantifying in vivo gene delivery.