Myostatin Gene Targeting In Cultured Han Yang Ovine Skeletal Mucle Cells

Gene targeting is a technique that integrates exogenous gene into the expected site of cellular chromosome by homologous recombination to change cell or animal's heritage characters. This paper report an investigation designed to knock out myostatin gene by gene targeting in Han yang ovine skeletal muscle cells. Two promoter-trap targeting vectors MSTN-GFP and MSTN-Neo were constructed and were used to transfect fetal and newborn ovine primary skeletal muscle cells. Both GFP expressing cells and drug resistant cells were obtained at an overall targeting efficiency of 6 × 10^-5 . G418 resistant cells were characterized by PCR and Southern blotting after growing into cell clones.Myostatin (MSTN), a member of the transforming growth factor- β (TGF- β ) superfamily, has been shown to be a negative regulator of myogenesis. The existing of a rare multiple birth Han yang sheep in China encourage us to explore the possibility of down regulation of the activity of myostatin gene by gene targeting whereby a sheep with merits of both multiple birth and well-developed muscle might be created.A 5.0kb and 1.0kb MSTN gene fragment was obtained by long PCR from genomic DNA preparations and was cloned into pMD-18T vector respectively. The promoter trap vector MSTN-Neo comprised a 5.0kb fragment of the MSTN gene containing exonl, intronl,exon2, intron2 and a portion of exon3.The long homologous arm was 4.6kb from the 5.0kb fragment; an 1.0kb fragment containing a portion of exon3 and the 3' end region of the MSTN gene. The short homologous arm was 0.8kb of the 1.0kb fragment. The skeletal bacterial cloning vector was pGEM-3Zf(-). The selection marker gene were Neo and GFP respectively. These two promterless targeting vector were constructed by inserting successively the three DNA fragments into the cloning vector. Linear vector DNA molecules were produced by cutting the vector at the unique AtII site and were used for transfection of ovine skeletal muscle cells in culture. The vector MSTN-GFP was constructed in a similar way except that the selection marker Neo was replaced by GFP.Primary Han yang ovine skeletal muscle cells were isolated from the hind limb of a fifty days old male fetus and of a one week old lamb respectively. The muscle tissue was disaggregated by trypsin solution and the cells were cultured and passaged in vitro. Ovine skeletal muscle cell line was constructed.Transfecting primary newborn ovine skeletal muscle cells with MSTN-GFP using lipofectAMINE according to the maufacturer's protocol. The expression of GFP in cells was confirmed by observing the green fluorescence 24hs after transfection. We could detected, on average, 2³ fluorescent cells per 10⁵ recipient cells after transfection and growth for 24 hours.48h after transfection ovine skeletal muscle cells and fetal skeletal muscle cells with MSTN-Neo, G418 selection was applied for 6¹0days. For the comparison of the effect of serum on drug selection 15% FCS and 10%FCS were used in the culture medium. A total of 88 Neo-resistant colonies were generated. Homologous recombination events were detected by PCR amplification and Southern blotting, only one clone was confirmed that one allele of myostatin was targeted.In this paper, we report cloning of Han yang ovine myostatin gene, construction of promoter trap gene targeting vectors, isolation and culture of ovine primary skeletal muscle cells, and production and characterization of targeted cells. A scheme for rescuing the targeted cells by nuclear transfer is under way.