Abstract
BackgroundThis 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.ResultsThree-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.ConclusionsWe 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.
Highlights
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
Since the single cell cloning became the obstacle of producing gene targeting clone, we tried to derive the transgenic cell lines from ovine satellite cells transfected with pEGFP-N1 plasmid as a model of transgenic satellite cell
Prior to utilization of fetal transgenic satellite cells for nuclear transfer, sex detection of transgenic cell lines isolated from single cell cloning is necessary because the gender of transgenic embryo can be determined by sex detection of nuclear donor cells
Summary
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. Satellite cells are a population of adult muscle stem cells that play a key role in mediating muscle regeneration These mononuclear cells are easy to obtain from in vitro culture, can be isolated with little harm to the structure and function of the tissues and organs and have strong proliferation capacities [1]. Several protocols have been established for sexing the embryos and cell lines in farm animals Among of these methods, PCRbased sexing assays are generally favored, because of the advantages of being relatively simple, rapid, and Salabi et al Journal of Biological Research-Thessaloniki (2014) 21:22 inexpensive [3,4]. We investigated in vitro culture and cell cloning of sheep satellite cells to establish a sheep cell line and to develop an ovine primary satellite cells sexing assay that was accurate, inexpensive and relatively fast. Our findings provide an experimental basis for the research and application of satellite cells in other fields, such as livestock breeding
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