Fig. 2

Vector construction and validation using transfection into PEFs. Construction of an inducible vector (FUW-tetO-pMYOD1) carrying the MYOD1 gene isolated from satellite cells in 3-day-old LYD biceps femoris. Cells were cultured in a serum-free basal medium containing 2% horse serum and 4 ng/mL doxycycline (DOX). a PCR results using genomic DNA (gDNA) from PEFs transfected with FUW-tetO-pMYOD1 to confirm the integration of the exogenous pMYOD1 gene. Cells were sampled on days 3, 6, and 9 of DOX treatment. The FUW-tetO-pMYOD1 plasmid was a positive control, and PEFs without transfection were the negative control. The arrows indicate the FUW-tetO-pMYOD1 plasmid vector and loading control pACTB (porcine beta-actin; internal protein). b Immunofluorescence images for pMYOD1 in PEFs transfected with FUW-tetO-pMYOD1. Scale bar = 100 μm. c qPCR results of PEFs transfected with the FUW-tetO-pMYOD1 vector to confirm the expression patterns of muscle-associated genes (Exo-MYOD1, Endo-MYOD1, PAX7, MYF5, and MYOG). Cells on day 0 were used as a negative control. Relative gene expression is represented as a trend line, describing 1 as the value of day 0 in Endo-MYOD1, MYF5, and MYOG and the value of day 3 in Exo-MYOD1 and PAX7