Fig. 4.

Expression analysis of IG transcripts. The position of the CLPG mutation is marked by the black arrow. The white arrows correspond to the positions of the TS2 and TS1 transcription start sites, respectively. MW, molecular weight marker. (A) Results of strand-specific RT-PCR experiments by using a 593-bp amplicon spanning the CLPG site and gluteus medius RNA from animals of the four possible CLPG genotypes at 2 weeks before and 8 weeks after birth. The amplicon was amplified from the cognate genomic DNA extracted from skeletal muscle as positive control, gluteus medius cDNA synthesized by using either one (specific for D←G transcripts) or the other (specific for D→G transcripts) primer, and RT-treated cDNA in the absence of primers. A 428-bp β-actin amplicon was amplified to control for the quality of the RNA. The CLPG amplicons were directly sequenced; the portions of the electropherograms spanning the CLPG site are shown, revealing the preferential expression of the CLPG allele in C^Mat/+ and +/C^Pat animals. (B) Results of RT-PCR experiments for 11 amplicons spanning the DLK1-GTL2 IG region (1′–11′ in Fig. 1) by using random primed gluteus medius cDNA (RP cDNA) from animals of the four possible CLPG genotypes at 2 weeks before and 8 weeks after birth. Amplicon 5′ is marked by an arrow as it spans the CLPG site. The same amplicons were amplified from the cognate genomic DNA (gDNA) and randomly primed cDNA with or without RT. The latter were all negative and are not shown. The cDNA amplicons were directly sequenced, and SNP markers in the region were used to determine the parental origin of the transcripts when possible. Biallelically expressed amplicons are marked by both a maternal (M) and a paternal (P) of equal size. Preferential expression of one allele is reflected by the relative size of the corresponding symbols. Monoallelically expressed amplicons are marked by M or P if the allele is madumnal or padumnal, respectively. In the absence of informative polymorphisms, the amplicons are unlabeled. (C) Representative results of PCR experiments performed with 47 overlapping amplicons spanning a 32-kb IG segment (Fig. 1) by using genomic DNA (gDNA) and random primed gluteus medius cDNA (RP cDNA) from a +/C^Pat fetus. Controls by using cDNA synthesized without RT were all negative and are not shown. (D) Results of strand-specific RT-PCR experiments performed with 11 amplicons, labeled A–H in Fig. 1 (i.e., A and A′ and B and B′, are distinct amplicons with virtually identical position) by using genomic DNA (gDNA), gluteus medius cDNA synthesized by using either one (specific for D←G transcripts) or the other (specific for D→G transcripts) primer, RT-treated cDNA in the absence of primers, and RT minus RNA with both primers.