Primary Culture Of Porcine Skeletal Muscle Satellite Cells And Chromosomal Localization, Spatio-temporal Distribution And Polymorphism Of The Porcine TRIM55 Gene

Skeletal muscle satellite cells are mononucleated myogenic cells, located between the sarcolemma and basement membrane of terminally-differentiated muscle fibres. In the normal course of events , muscle satellite cells are quiescent, but they can be activated when the muscle fiber harmed by any form of trauma, damage or injury. The activated satellite cells can proliferate, and then the daughter cells migrate to the damaged area of muscle tissue, or they fuse with the existing muscle fiber, donating their nuclei to the fiber, which help to regenerate the muscle fiber. Because of the key role played in skeletal muscle growth, development, and regeneration, the satellite cell is research hotspot at present.Pig breeding is always focused on improving lean percentage and meat quality from the past through the present to the future. The growth and development of skeletal muscle play a crucial role in the improvement of the lean percentage and meat quality. In order to obtain insights into the growth rules, structural alterations of myocytes and the function of genes involved in muscle development, the skeletal muscle cells were primarily cultured. And the primary culture of skeletal muscle satellite cells provides a new outlet as cell model to do the research while the work is hard completed in vivo.In this study, the satellite cells were isolated from longissimus dorsi muscles of 1 day-old piglet. Muscles were ground and digested with Collagenase II. The Preplating technique was used to remove non-myogenic cells from satellite cells suspensions. The results showed that this method could obtain highly purified satellite cells and these cells could be promoted proliferation and differentiation, which can be used for in vitro study the growth and development of myocytes.Establishment of in vitro primary culture of porcine skeletal muscle satellite cells can provides a platform for experimental operation of satellite cells, as well as lays a solid background for studying the function of genes related to development of skeletal muscle. The RING finger proteins function in a variety of fundamental cellular processes such as signal transduction, ubiquitination, gene transcription, differentiation and morphogenesis. Recently, a novel family of muscle-specific RING finger (MURF) proteins was identified in mouse and human which was a special subgroup of the RING Finger-B box-coiled-coiled (RBCC) proteins. Tripartite motif=containing 55 (TRIM55) gene (also know as RNF29, MURF=2) is a member of the MURFs family. Several recent reports demonstrated that TRIM55 acted as cytoskeletal adaptors and signaling molecules by associating with myofibril components (including the gaint protein, titin), microtubules and/or nuclear factors and was also involved in maintenance of the precise interactions and alignment of complex cytoskeletal networks. Therefore TRIM55 is proposed to play an important role in growth of myocytes. In this study, TRIM55 gene is selected to be researched as a candidate gene which impacting skeletal muscle development in the pig. It would lays an important basis on further functional study of the TRIM55 gene on the impaction of skeletal muscle development through its isolating, chromosomal localization, spatio=temporal distribution, SNP detection, and association with pig production traits, providing a molecular genetic foundation on its application in the molecular marker assisted selection (MAS) in the future. The main results are showed as follows:1. The human TRIM55 cDNA was used to search in the 'EST=other' database with the standard BLAST algorithm for homologous pig expressed sequence tags (ESTs). Five partially overlapping porcine ESTs were searched and assembled to produce a pig EST=contig. Two primer pairs were designed from the pig EST contig. By the primers, two fragments in 3'=untranslated region (3'-UTR) of pig TRIM55 gene were obtained (GenBank accession number are DQ 160212 and DQ 160211).2. The INRA=University of Minnesota porcine radiation hybrid (IMpRH) panel was employed for detecting the precise localization of TRIM55 gene. The data of PCR results was analyzed using the mapping analysis software available on the web server. The statistical analysis revealed that SW1475 located on chromosome 4 (SSC4) is TRIM55's closest linked marker (LOD=9.74), and the other three microsatellite markers linked with TRIM55 were SW839 (LOD=8.53), SW742 (LOD=7.21) and SW1073 (LOD=6.2). SW839 was previously mapped to 4q11-14. Therefore the most probable chromosomal localization for TRIM55 is SSC4q11-14. 3. The semi-quantitative RT-PCR was performed to determine the tissue distribution of TRIM55 gene in five different tissues from 1 day-old Chinese Tongcheng porcine heart, skeletal muscle, spleen, liver, and kidney. The skeletal muscles from embryos (30d, 60d, 90d) and postnatal pigs (1d, 28d, adult) were used for gene temporal expression analysis. The RT-PCR results revealed that the porcine TRIM55 was restrictedly expressed in heart and skeletal muscle, although a low level of transcripts was detected in spleen, and that TRIM55 expression levels were up-regulated in pig skeletal muscle during embryo stage, but down-regulated during the pig postnatal development.4. Direct sequencing of PCR products revealed two single nucleotide polymorphisms (SNPs) in 3'-UTR of TRIM55 gene. The A/G transition at position 162 can not be recognized by any restriction endonucleases and the other single nueleotide mutation (C/T) at position 213 spans a Bsh1236Ⅰrestriction site. PCR-RFLP technique was employed to detect the C/T mutation and the polymorphism was analyzed among the different pig groups. The allele frequencies and the allele distribution were determined among these populations.5. The Bsh1236Ⅰ-RFLP of TRIM55 gene was genotyped in the experimental pig populations constructed under the cooperation of our lab and animal husbandry bureau of Tongcheng county, including Landrace, Large White, Landrace♂×(Large White×Tongcheng)♀and Large White♂×(Landracex Tongcheng)♀. The General Linear Model (GLM) was used to carry out association analysis. The preliminary results indicated that the C/T mutation showed a significant association with the average daily gain from birth to market, shoulder fat thickness and average fat thickness (P=0.0363, 0.0401 and 0.0473, respectively).