Isolation of a potential gene for obesity in a transgenic mouse
The objective of this research was to identify the genomic segments flanking the transgene LTR-tat3 in obese mouse, based on the hypothesis that the obese phenotype is the result of the genomic alteration caused by the integration of a transgene LTR-tat3. Two PCR strategies were applied to amplify the mouse genomic sequences flanking the LTR-tat3 insert. The inverse PCR approach was tried but was not successful. This may be due to the inappropriate DNA fragment sizes produced by the restriction digestion or incomplete digestion. The arbitrary PCR approach was then utilized to amplify the flanking sequences. For the left junction of LTR-tat3 insert, a set of twenty 10-mer arbitrary primers (OPA) were each used separately in conjunction with a specific primer complementary to the LTR sequence. Of the 20 OPA primers, nine produced positive bands with Southern blot analysis. These nine positive bands are potentially target sequences flanking LTR-tat3 insert by virtue of the tandem array of multiple LTR-tat3 transgene. For the right junction of LTR-tat3 insert, a modified arbitrary CR--TAIL--PCR method was applied using three nested specific primers with a set of arbitrary degenerate primers (AD) as well as interspersion of asymmetric and symmetric PCR cycling to geometrically favor amplification of the desired target sequence over nonspecific products. Of the four AD primers, the AD1 primer produced a band of 1100 bp that hybridized with the LTR-tat3 specific DNA probe. These probe positive fragments can be used as templates for making new specific primers for further chromosomal walking to identify additional sequences adjacent to the transgene.