According to the previous reports [4,5], QM7 cells (American Type Culture Collection, Manassas, VA, USA) were maintained at 37[degrees]C in an atmosphere of 5% C[O.sub.2] and 60% to 70% relative humidity with Medium 199 containing 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 2% chicken serum (Sigma- Aldrich, St.
Total mRNAs were isolated from the differentiated QM7 cells during 3 days of myotube differentiation as well as the undifferentiated QM7 cells, and all of samples were triplicated.
To validate the expression patterns of calcium-related genes, total mRNAs were isolated from the undifferentiated QM7 cells and differentiated QM7 cells (day 1 to day 3 of differentiation periods).
Total RNA from undifferentiated regular QM7 (rQM7) and SH3RF2 KO QM7#4 cells was isolated using TRIzol Reagent (Invitrogen, USA) according to the manufacturer's instructions.
After transfection of the Cas9-SH3RF2 gRNA expression vector into QM7 cells, GFP-positive cells were identified by FACS and a single cell was isolated followed by in vitro expansion.
Regular QM7 (rQM7) and SH3RF2 KO QM7#4 cells had a similar morphology in the undifferentiated state (Figure 4A).
After transfection of the Cas9-MyoD gRNA expression vector into QM7 cells, GFP-positive cells were identified by FACS and finally a single cell was isolated followed by in vitro expansion (Figure 1C).
In the undifferentiated state, the expression of paired box 7 (Pax7), a critical marker of undifferentiated myoblasts, in regular QM7 (rQM7) and MyoD KO QM7#4 cells was compared by qRT-PCR but no significant differences were apparent between them (Figure 3A).
Global gene expression analysis by RNA sequencing in QM7 MyoD KO cells
The percentage of GFP-positive cells in the cumate-GFP QM7 cells also increased depending on the concentration of cumate (Figure 2B).
However, cumate-GFP QM7 cells exhibited low expression of the GFP transgene in the absence of cumate, indicating that control of transgene expression using the cumate-inducible promoter differs among different types of avian cells (Figure 1).
Controlled Inducible transgene expression In chicken DF1 and quail QM7 cells.