RT-PCR and Southern hybridization analysis of TrfmRNA.
The cartoon in the top right corner illustrates the placement of primers in the Trf gene, which were used for RT-PCR. For each animal, lane 1 represents full-length RT-PCR, lane 2 represents RT-PCR across the splice junction for intron 15, and lane 3 represents RT-PCR across the splice junction for intron 16. The agarose gel in the top panel was analyzed by Southern blot analysis. Probe A is specific for cDNA reverse transcribed from correctly spliced Trf mRNA, completely excising intron 16. Strong hybridization signals are shown in lanes from animals carrying a wild-type allele and are absent inTrfhpx/hpx mice. Probe B hybridizes to cDNA reverse transcribed from Trf mRNA resulting from use of the cryptic splice donor site in exon 16, causing excision of intron 16 plus 27 bp of exon 16. Probe B hybridizes specifically to RT-PCR products from the Trfhpx allele.
