Previous studies have shown that VGLL2, a member of the mammalian Vestigial-like (VGLL) family, plays important roles in the growth and development of animal skeletal muscle, but its specific role in the development of chicken skeletal muscle is unclear. The main goal of this study was to explore the biological functions of VGLL2 in the development of chicken skeletal muscle and the proliferation and differentiation of skeletal muscle cells in vitro. In this study, we detected the effect of VGLL2 on the proliferation of myoblasts by CCK8, EdU and flow cytometry analyses after overexpressing and interfering with VGLL2. Indirect immunofluorescence was used to detect the effect of VGLL2 on the differentiation of myoblasts. qRT-PCR and hematoxylin and eosin (H&E) staining were used to evaluate the effects of VGLL2 overexpression on the growth rate and muscle fiber structure of chicken skeletal muscle. The results showed that VGLL2 inhibited the proliferation of primary cultured chicken myoblasts and promoted the differentiation of these cells. Interestingly, food intake and muscle fiber development were significantly enhanced by the overexpression of VGLL2 in chickens. Taken together, these data demonstrate that the VGLL2 gene may be a useful marker for improving muscle mass in poultry.

Skeletal muscle satellite cells are stem cells characterized by their multipotency and capacity for in vitro proliferation. However, primary skeletal muscle satellite cells demonstrate limited proliferative capacity in vitro, which impedes their investigation in poultry skeletal muscle research. Cell immortalization techniques have emerged as valuable tools to address this limitation and facilitate the study of skeletal muscle satellite cell functions. This study achieved the immortalization of chicken skeletal muscle satellite cells through the transduction of primary cells with TERT (telomerase reverse transcriptase) amplified from chicken (chTERT) using a lentiviral vector via telomerase activity reconstitution. While the cells successfully overcame replicative senescence, complete immortalization was not achieved. Initial functional characterization revealed that the proliferative properties and cell cycle characteristics of the immortalized chicken skeletal muscle satellite cell lines (ICMS) closely resembled those of chicken primary muscle satellite cells (CPMSCs). Serum dependency analysis and soft agar assays confirmed that ICMS did not undergo malignant transformation. Furthermore, induced differentiation experiments demonstrated preserved differentiation capacity in ICMS. The established cell lines provide a fundamental framework for developing immortalized poultry cell lines and offer a cellular model for investigating poultry skeletal muscle-related functional genes.