A Preliminary Study On The Knockout Myostatin And Knockin MAAT In Sheep Somatic Cells

Gene targeting is a useful protocol to modify the cell genome based on the homologous recombination. According to the successful experiences on transgenic animal production and animal cloning, gene targeting could be utilized in many fields including theory and application research on genomic modification (such as site-specific gene repair, genetic defect therapy, gene knockout, infaust gene modification, even the site-specific integration), improving expression level of specific gene in animal mammary gland bioreactors, and promoting the industrialization of transgenic animal production. Therefore, the present study was undertaken to explore the possibility of gene targeting in sheep fibroblasts by using myostatin as targeting gene, mAAT as the integrated gene and Neo as mark gene.Firstly, 1.4kb and 4.3kb homologous arms were amplified from the female Dorset sheep genome by the long fragment PCR technique. The 4.3kb homologous left arms contains the exon I , II and part of intron II fragment, while the 1.4kb homologous right arms contains the exonIII and partial 3' region of myostatin gene. These two fragments were cloned into the pMD-18T vector for the sequencing analysis. Homologous sequence alignment indicated 100% homology with the ovine and 95% homology with bovine. These dataproved that the amplified fragment was the ovine myostatin gene, which could be used for the gene targeting research.Positive and negative selection vector pLoxP-1.4K-4.3K was constructed by linking 1.4kb and 4.3kb fragment into the pLoxP vector. Additionally, an insertion vector pM-AAT was constructed by cloning the mAAT gene into the modified pLoxP-1.4K-4.3K vector, downstream of the neo gene and upstream of the right homology arms. The PCR and restriction enzyme digestion result indicated that the anticipated construction was correct.Eight sheep fetal fibroblast lines and four female adult Dorset ear fibroblast lines were established by trypsin digestion and tissue attached culture method respectively. Four of eight fetal fibroblast lines were identified to be female and four were identified to be male by the SRY-PCR method. Chromosome analysis was performed in different cell passages. In our in vitro culture system, the fetal fibroblast could survive for 35 passages, and the adult Dorset ear fibroblast could survive for 25 passages, which might fill into the requirement of cell transfection and drug selection procedure for gene targeting research.Transfection and drug resist clone culture were performed by electroporation and lipofectamin-mediated DNA transfection methods with two kinds of vectors and different cell lines. In calculating the absolute transfection efficiency, Lipofect transfection method is better than the electroporation for the smaller DNA fragment; while electroporation is moresuitable for the larger DNA fragment. Three of neomycin resist clones transfected with pLoxp-1.4K-4.3K were identified to be positive by PCR examination, while no cell clones transfected with pM-AAT were identified to be positive, and only the mAAT was found to integrate into the genome chromosome. One hundred and five embryos derived from positive cell clones were transferred into 7 foster mothers. Among them, 2 recipients returned to estrus in the second estrous cycle, and one returned to estrus in the third estrous cycle. These results indicate that embryos derived from transfected cells could implant following transfer to recipients.The above research results indicate that gene targeting can be used to modify the genome of sheep fibroblast lines. However, more experiments should be performed to improve the in vitro culture conditions, gene targeting efficiencies, and single clone propagation.