The Establishment Of Mammary Epithelial Cell Line And HRM Genotyping Method On Myostatin Gene G.+6723 Loci Of Dairymeade Sheep

Dairy Meade sheep is a newly introduced dairy and meat dual purpose sheep breed of China,which has excellent performance both in lactation and mutton production.Dairy Meade sheep can be used as a potential breeding material for creating the new dairy or dairy/meat dual purpose sheep breed that fit the weather and feeding conditions of Inner Mongolia,China.The revealing of its genetic mechanisms related to dairy and mutton production performance could provide important theoretical support for the breeding process.Current study focused on the establishment of mammary epithelial cells of Dairy Meade sheep.First,the primary mammary epithelial cells of Dairy Meade sheep were isolated and cultured by tissue explants adherent and differential digestion method.The cell lines with SV40 LT gene stable transfection were obtained,which SV40 LT gene was transfected into primary mammary epithelial cells mediated by retrovirus to primary mammary epithelial cells.The primary mammary epithelial cells and SV40 LT transfected mammary epithelial cells were identified by morphological observation,growth curve measurement,immunofluorescence detection,marker gene expression detection and karyotype analysis.The results showed that the two kinds of cells all demonstrated typical"paving stone"or"polygonal morphology".Their cell growth curves were consistent with the general biological law and their chromosome were normal.The two cell line all stably expressed CK7,CK8 andβ-casein genes and immunofluorescence assay showed they expressed CK7 protein.Moreover,the SV40LT transfected cells could be cultured for 20 passages and still in good conditions.We also compared the growth traits and slaughter performance of Dairy Meade×STH（Small Tail Han sheep）F1 rams with AA and GG genotypes at loci g.+6723 of MSTN gene,and established a rapid genotyping method for this loci based on HRM（High Resolution Melting curve）technique.The results showed that there was no significant difference in the growth traits between AG and GG genotype rams.However,the dressed rate and net meat rate of AG rams was significantly higher than GG rams.Through the optimization of primer pairs,Mg²⁺concentration（4.0 m M）,primer concentration（0.2μm）and template concentration（0.25 ng/μL）of HRM reaction system,a rapid genotyping method for this loci was finally established.In the study,we established the stabled transfection mammary epithelial cell line of Dairy Meade sheep,which provided an ideal experimental material for the study of its lactation regulation mechanism in the future.The establishment of rapid genotyping method of MSTN gene g.+6723 loci based on HRM technology provided technical support for the application of this SNP in breeding process.