鸡骨骼肌卫星细胞系的建立及分析

doi:10.11843/j.issn.0366-6964.2023.12.009

Abstract

Abstract: The purpose of the study was to establishment of chicken skeletal satellite cell line by constructing SV40-LT gene with lentivirus and transforming primary chicken skeletal satellite cell line (PMSCs). In this study, chest tissues from 20-30 healthy AA chickens of 15 embryonic ages were collected, and a mixed enzyme digestion method was used to isolate and cultivate PMSCs. Randomly divided the cells into two groups, each with three replicates. The treatment group was transfected with SV40-LT lentivirus for 48 hours, and then replaced with medium containing 1 μg·mL^-1 puromycin for screening and cultivation based on the treatment group and the control group (virus not transfected). After all cells in the control group died, the cells in the treatment group were continuously subcultured to obtain a chicken skeletal muscle satellite cell line. Immunofluorescence assay was used to analyze the expression of the marker gene PAX7 in chicken skeletal muscle satellite cells; Cell proliferation characteristics were detected using CCK-8 and cell cycle analysis; Serum dependence and soft agar analysis were used to detect its malignant transformation status; Constructing an induced differentiation model to test its ability to induce differentiation. The 92% of cells in PMSCs were positive for the PAX7 gene, which could be used for subsequent research. Compared with primary chicken skeletal muscle satellite cells, SV40-LT was expressed 15-fold in chicken skeletal muscle satellite cell lines (IMSCs P12), and after continuous passage to 12th generation, both IMSCs P12 and PMSCs exhibited fibrous morphology. IMSCs P12 showed higher proliferative activity and did not undergo malignant transformation. Subsequently, IMSCs P12 was induced to differentiate. During the stage from 70% proliferation to 1 day of induced differentiation, the expression of PAX7 genes in IMSCs P12 and PMSCs was downregulated, while the expression of MyoD and MyHC genes was upregulated. This study successfully cultivated chicken skeletal muscle satellite cell line by transfecting the SV40-LT gene, which has similar characteristics to the primary chicken skeletal muscle satellite cells. The establishment of this cell line provided a new platform for studying functional genes related to poultry skeletal muscles, and also laid the foundation for the study of SV40-LT gene immortalization in poultry cells.

Key words: chicken, skeletal muscle satellite cells, SV40-LT, cell line, PAX7

CLC Number:

S831.2

WANG Yanxing, ZHANG Yushi, JI Haigang, LIU Yang, NIU Yufang, HAN Ruili, LIU Xiaojun, TIAN Yadong, KANG Xiangtao, LI Zhuanjian. Establishment and Analysis of the Chicken Skeletal Satellite Cell Line[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 4972-4981.

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Establishment and Analysis of the Chicken Skeletal Satellite Cell Line

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