Background: This study was performed to describe the basic methods to isolate and culture of primary satellite cells (PSCs) obtained from 50 to 60-day-old sheep fetuses, single cell cloning of transfected PSCs and sexing of ovine PSCs based on the ZFY/ZFX, amelogenin and high-motility-group (HMG) box sequences.
Results: Three-step enzymatic digestion method increased PSCs isolation from tissue and reduced the damage of cells during long time incubation with enzymes. The results of cloning showed that the 103 and 81 clones (from a total of 184 clones) were derived from feeder and bFGF treatment, respectively. The overall sexing efficiency in the present study was 100%. Southern blot results of sex determination were in complete agreement with PCR-amplified bands which confirmed that the HMG box of SRY gene amplified from the ovine genome and that was specific for male.
Conclusions: We successfully isolated and cultured sheep primary satellite cells via mechanical and enzymatic disaggregation. Our finding demonstrated that use of feeder and addition of bFGF to the culture medium improved cloning efficiency. The results of sex detection demonstrated that these methods can be applied to detect the sex of primary satellite cells and to determine the sex of sheep embryo prior to produce sheep embryos by somatic cell nuclear transfer technique in vitro. Nevertheless, our findings suggested that sex determination of satellite cells base on amelogenin sequence can be accurate, relatively simple, rapid, and inexpensive.
Keywords: Enzymatic disaggregation; Satellite cells; Sex determination; Single cell cloning.