Production Of Myostatin Gene Knock-Out Cattle By Somatic Cell Nuclear Transfer

Myostatin (MSTN) is a specific negative regulate-factor for the growth and development of mammalian skeletal muscle. Reduction or loss of MSTN function resulted overgrowth of animal muscle. The natural mutation of bovine MSTN gene leads to the phenomenon of "double muscle". Compared to nature hybridization, gene targeting will take short breeding cycle to produce trangeneic animal which has stable genetic characters. The gene targeting vectors were constructed for homologous replacement of an endogenous MSTN allele. Bovine fetal fibroblast cells were used for transfection and screening followed by somatic cell nuclear transfer with MSTN knockout positive cells as donor cells. The constructed embryos were transfered into recipients and resulted in preganencies. The results will allow the production of gene-knock livestocks and research of MSTN gene functions.1. Construction of knock-out vectors for bovine MSTNTwo MSTN gene targeting vectors (pPLP-MSTN and PⅢ-MSTN) were constructed. The common homologous recombination targeting vector pPLP-MSTN included 2.8kb short arm,4.0kb long arm and Ned gene with PGK promoter. The promoter trap targeting vector PⅢ-MSTN included 1.3kb short arm,6.8kb long arm, EGFP gene without promoter and Neor gene with PGK promoter as marker genes for double positive selection. The homologous arms of two vectors were derived from ear tissue of Japanese black cattle.2. Preparation of MSTN gene knock-out bovine fetal fibroblast cellsThe linearized pPLP-MSTN was introduced into fetal fibroblast cells of Dalian Xuelong black cattle, Japanese black cattle and Luxi cattle by electroporation.363 clones were obtained after selection with G418 and none of MSTN gene knock-out clone was found. The linearized PⅢ-MSTN was introduced into fetal fibroblast cells of Japanese black cattle and Luxi cattle by electroporation.104 and 164 clones were obtained respectively after selection with G418.20 and 29 clones expressing green fluorescence were observed under microscope. In 20 clones of Japanese black cattle, one was found to have undergone the desired recombination event by PCR analysis and PCR product sequencing. Therefore, the relative targeting frequency was 5.00% (1/20), and the absolute targeting frequency was 0.27×10-7(1/3.4×107). In 29 clones of Luxi cattle, none was found to have undergone the desired recombination event.3. Preparation and transfer of MSTN gene knock-out bovine embryosMSTN gene-targeted bovine fetal fibroblast cells were recruited as the nuclear donor cells, and enucleated oocytes were applied as the recipient cytoplasm.370 matured oocytes were enucleated and inserted donor cell, and 363 of them were exposed for electronic pulse.259 (71.3%) of them were fused. All reconstructed embryos were activated and cultured in vitro, and 66 (25.5%) of them developed to blastocyst stage. The 18 blastocysts were transferred into 12 uterus, and 3 (25%) uterus pregnancies.