Gene expression profiling of hypertrophied muscles in callipyge sheep.

摘要

Callipyge sheep exhibit extreme postnatal hypertrophy in the loin and hindquarters as a result of single nucleotide polymorphism (SNP) in the imprinted DLK1-MEG3 gene cluster on ovine chromosome 18. The callipyge SNP up-regulates the expression of nearby imprinted transcripts when inherited in cis. This thesis contains phenotypic measurements of lambs possessing a callipyge allele from 10 days of age to 200 days of age. These animals were used in three studies designed to profile gene expression in lamb muscles to determine down-stream effects of paternal allele specific and maternal allele specific transcripts. Affymetrix Bovine Expression Arrays and quantitative PCR were used to discover transcripts differentially expressed as a result of the callipyge mutation. To determine the transcripts affected during callipyge muscle hypertrophy, two hypertrophied muscles (longissimus dorsi and semimembranosus) from callipyge (+/CPat n=8) and wild-type lambs (+/+ n=8) were compared across four ages that encompass the postnatal muscle hypertrophy development. Analysis methods by MAS5 and RMA identified 149 to 378 transcripts, respectively, that were differentially expressed in callipyge muscles. These included metabolic enzymes, apoptotic factors, proteins involved in regulating common signaling pathways, and transcription factors. Thirty-three novel transcripts were validated by qPCR to be differentially expressed between callipyge and normal muscles across additional ages. The third experiment aimed at detecting differences in genes expression affected by the presence of a maternal callipyge allele (CMat/+ and C/C). These lambs were contrasted against lambs with a wild-type maternal allele (+/ CPat and +/+). The microarray and qPCR data only validated significant differences in expression of MEG3 and MEG8, both genes within the callipyge imprinted cluster. These data indicate that the up-regulation of MEG3 and MEG8 does not influence gene expression of any transcripts on the bovine expression array which are not located near the callipyge mutation. High levels of DLK1 and RTL1 in the paternal heterozygous lambs are enhancing factors involved in existing muscle growth mechanisms including the AKT/mTOR signaling pathway.