Figure 4

Identification of porcine MSTN gene regulatory elements by ABI-REC. (A) Genomic structure and reporter design of porcine MSTN gene regulatory elements. Promoter regions 3.8 kb and 2.3 kb in size were fused into a pGL3-basic plasmid, and a 1.4 kb terminator region was fused into a pGL3-promoter plasmid. (B) Asymmetric PCR reactions were used to generate the fused fragments of three sequences stated above. Here, template amounts were assessed at 100 ng and 10 ng. Red arrows denote the fused fragment. (C) The PCR products in (B) were treated as indicated in “Methods”. Single colonies were selected and sequenced. Digestion mapping indicated that the inserts had been successfully fused into target plasmids. (D) The transcriptional activity of the cloned porcine MSTN gene regulatory elements. Equal molar quantities of all these constructs were transfected into mouse myoblast C2C12 cells, and then the luciferase level was measured under either proliferating or differentiating conditions. pRL-TK plasmid that expresses Rluc was co-transfected as internal control. Relative luciferase activity was calculated as ratio over that observed in control transfections, where Fluc activities were normalized to Rluc activities. Error bars indicate mean ± SD from three independent transfections, each in triplicate. The inset indicates proliferating and differentiating C2C12 cells. (E) Function of MSTN elements with respect to expression of the reporter gene. The MSTN promoter, EGFP CDS, and MSTN terminator were assembled as an expression cassette by ABI-REC, and their expressivity was assessed by transfecting it into C2C12 cells. This expression cassette is capable of driving EGFP expression efficiently, in comparison to positive control pIRES2-EGFP. Exposure time, 0.5 s; scale, 10 μm. (F) Quantitation of EGFP intensity in (E) by ImageJ. Five randomly captured EGFP clips in each transfection were analyzed by ImageJ to calculate the counting area of fluorescent cells as an indicator of EGFP expression level (Green/total × 100%). The threshold was set between 88-225 pixels. (CMV-EGFP-SV40 polyA) cassette of pIRES2-EGFP is the positive control to pig MSTN (promoter-EGFP-terminator) cassette. This result implies that the two identified MSTN elements are able to work coordinately to regulate gene expression. This cassette could be used to control transgene expression and to transfer genes of interest to endogenous sites on the pig MSTN locus.