Fig. 1
From: MSTN knockout enhances the production of MYOD1-mediated steak-type cultivated meat
MYOD1-induced myogenic conversion of bovine fibroblasts using piggybac transposon. A Structure of MYOD1-expression vector with ubiquitous promoter (i) and experimental scheme (ii). B Immunofluorescence images using anti-desmin antibody in cells transfected with ubiquitous MYOD1 expression. NC = Un-transfected negative control, 1st (-) = Transfected vector, but stained without the primary antibody, Vector = Transfected with MYOD1 vector. Green = GFP, Red = Desmin, and Blue = Nuclei. Scale bar = 150 μm. C Bright and fluorescent images of GFP reporter expression on day 9 after transfection before (i and ii) and after passaging (iii and iv). Scale bar = 275 μm. D Structure of conditional MYOD1 expression vector (i) and experimental scheme (ii). 5′ ITR to 3′ ITR sequences were all packaged in single vector. E Immunofluorescence images using anti-desmin antibody. Cells integrated with vectors were sorted using RFP reporter and divided into following three groups. Dox- = untreated with Dox and stained using all antibodies, 1st (-) = treated with Dox and stained without using the primary antibody, Dox+ = treated with Dox and stained using all antibodies. Green = GFP, TxRed = Desmin, and Blue = Nuclei. Scale bar = 275 μm. F Semi-quantitative real time PCR analysis of MYOD1 and other myogenesis related factors, related to homeobox gene. Cells transfected with piggybac vectors were sorted using reporter, and 2 μg/mL of doxycycline was treated for 7 d before analysis. Data are presented as mean ± SD (n = 3). Asterisks indicate statistically significant differences (****P < 0.0001; n.s., not significant). G Structure of the dox-dependent MYOD1 expression vector with puromycin resistance gene (H) immunofluorescence images of trans-differentiated primary bovine fibroblasts using MYOD1-puro vector. Dox- = untreated with doxycycline; Dox+ = treated with doxycycline. Green = immunofluorescent stain, Blue = Nuclei. Scale bar = 150 μm. I Semi-quantitative real time PCR analysis of MYOD1 and other myogenesis markers, related to homeobox gene in trans-differentiated primary cells. Data are presented as mean ± SD (n = 3). Asterisks indicate statistically significant differences (**P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant). J immunofluorescence images of trans-differentiated bovine immortalized fibroblasts using MYOD1-puro vector. Dox- = untreated with doxycycline; Dox+ = treated with doxycycline. Green = immunofluorescent stain, Blue = Nuclei. Scale bar = 150 μm. K Semi-quantitative real time PCR analysis of MYOD1 and other myogenesis markers, related to homeobox gene in trans-differentiated bovine immortalized cells. Data are presented as mean ± SD (n = 3). Asterisks indicate statistically significant differences (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant)