Blocking Of Mstn Expression By Rnai And Gene Targeting In Goat Somatic Cells

Myostatin?MSTN?,a growth and diferentiation factor-8?GDF-8?,is a member of the transforming growth factor-??TGF-??superfamlly.Myostatin null mice and naturally myostatin mutant double muscling cattles show that MSTN was an inhibitor of skeletal muscle growth.RNA interference?RNAi?is a phenomenon of sequence-specific post-transcriptional gene silencing?PTGS?initiated by double-stranded RNA?dsRNA?.Now it has been used as a new method to investigate corresponding gene function and regulation of gene expression.Gene targeting is a powerful technology for transforming genetic information of the organisms directionally.Traditional gene targeting is derived from homologous recombination?HR?between exgenous gene and cellular chromosome and mediated by embryonic stem cells?ESC?or fetal fibroblasts.Through specially mutating the gene of the animals,we can set up animal models of human diseases and investigate the function of the gene.In this article,we selected the caprine MSTN as the targeted object and blocked its expression by RNAi or gene targeting technology respectively.Firstly,four RNAi-vectors expressing candidate miRNA targeting MSTN cDNA sequence and a vector overexpressing MSTN were constructed.Then each of the RNAi-vectors and overexpressing vector were cotransfected into 293FT cells and screened respectively.Two most effective miRNAs were selected and ligated into miRNA expression vectors marked with GFP,and then transfected into GFF and SMSC cells to test inhibitory activity separately.Moreover,interferon?IFN?response genes,such as IFN-?,OAS2,Mx1,IFI44 and STAT1,were examined for their expression.Furthermore,we took use of gene targeting technology to construct the MSTN knockout vector and transfected it into GFF cells.Resistant cell clones were selected with drugs and identified to obtain positive cell clones and be used as the donor in downstream nuclear transfer.The study was divided into five parts.The main results were as follows:1.We constructed miRNA RNAi-vectors against caprine MSTN and examined their efficacies by image analysis,qPCR and western blot analysis.Firstly,four miRNAs of caprine MSTN were designed using an online tool named as Block-iT RNAi Designer.Individual miRNAs were converted to intramolecular stem-loop structures known as pre-miRNAs.We annealed and cloned the oligonucleotides encoding the engineered pre-miRNAs into pcDNA6.2-GW/miR,to generate pD-miRNA plasmids.In addition,full-length caprine myostatin was also chemically synthesized and cloned into pEGFP-C1,to generate a C1-MSTN plasmid expressing a myostatin and EGFP fusion protein,for monitoring myostatin protein expression.For transfection,individual pD-miRNA plasmids were co-transfected with Cl-MSTN into 293FT cells at a ratio of 3:1.Twenty-four hours after transfection,293FT cells transfected with Cl-MSTN were photographed using a fluorescence microscope.All settings for image capture were kept consistent.Cells fluorescence in the images was measured and quantified using IPP software.Averages of fluorescence intensity represent levels of myostatin-EGFP fusion protein.Forty-eight hours after transfection,cells were harvested and examined for myostatin expression.To determine myostatin expression,one half of the cells were used for qPCR and the other half were used for western blot analysis.The results showed that all miRNAs caused significant downregulation of myostatin expression compared to miRNA-NC.miRNA-1 and miRNA-3 showed the highest efficacy,reducing myostatin mRNA by up to?3 8%and myostatin protein by?48%,respectively.Our results demonstrated that specific and efficient silencing of caprine myostatin can be achieved using miRNA.miRNA-1 and miRNA-3 were selected as the optimal miRNAs to knock down endogenous myostatin in subsequent experiments.2.Efficient MSTN gene silencing was observed in both transiently and stably transfected GFF cells,along with off-target effects against all plasmid constructs.GFF cells were isolated by attaching tissue explants from a 60-day-old goat fetus and purified by trypsin.GFF cells were examined by cell morphology,identification of sex-determined region Y gene?SRY?,growth curve and transfection efficiency,which indicated that it's suitable for the need of transgenic clone.The optimal concentration of Blasticidin?Bla?was determined to 10?12g/mL in the cell resistance experiment.miRNA-1 and miRNA-3 oligos were cloned into pcDNA6.2-GW/EmGFP-miR to generate pDG-miRNA.The miRNA oligos were inserted into the 3'-UTR of the EmGFP gene,for monitoring miRNA expression.The ability of the pDG-miRNA plasmids to produce RNAi effects was tested in transiently transfected GFF cells.The results showed that both miRNA-1 and miRNA-3 could significantly inhibit MSTN expression via cleavage of their target sequences.At the same time,we discovered that the expression of IFN-stimulated genes?IFN-? and OAS2?increased sharply.The ability of RNAi to silencing MSTN expression was also tested in stably transfected cells.The results showed that only miRNA-1 could significantly inhibit MSTN expression,and the IFN response was much weaker in stably transfected cells than in transiently transfected cells.These results indicated that keeping miRNA doses well below the point at which the IFN response downregulates MSTN mRNA levels can ameliorate these off-target effects.3.Silencing of MSTN and off-target effects induced by miRNAs were detected in transiently transfected SMSC cells.SMSC cells were derived from vastus lateralis co-digested with Type 1-A collagenase and trypsin,and purified by differential adhesion.Activated SMSC cells in vitro culture eventually differentiate and fuse to form multinucleated myotubes.So cells in primary culture have to be passaged within forty-eight hours and cultured in a low density.The ability for SMSC cells to differentiate into muscles were identified by formation of myotubes,and the purity of cells were identified by immunocytochemistry of desmin.SMSC cells were examined by cell morphology and 70%transfection efficiency was achieved,which indicated that it's suitable for the need of gene targeting.To further test inhibitory activity,we transfected pDG-miRNA into SMSC cells transiently.The results showed that both miRNA-1 and miRNA-3 could significantly inhibit MSTN expression,and the expression of IFN-stimulated genes?Mx1,IFI44 and STAT1?also increased significantly.Our results indicated that miRNAs had triggered off-target effects,due to induction of IFN response genes,in transiently transfected SMSC cells.4.The gene targeting vector for goat MSTN was constructed,in which the validity of marker genes was also tested for positive and negative selection.Firstly,5.2kb and 1.1 kb homologous arms were amplified from the goat genome by the long fragment PCR technique.The 5.2kb 5' homologous arm is the fragment between 5' region and intron ?,while the 1.1kb 5' homologous arm contains the partial exon ? and 3' region of MSTN gene.These two fragments were cloned into the pMD-19T vector for the sequencing analysis.Homologous sequence alignment indicated 99.7%homology with goat and 95%homology with bovine.These data proved that the amplified fragments were the goat MSTN gene,which could be used for the gene targeting research.Positive and negative selection vector ploxPneo-MSTN was constructed by linking 5.2kb and 1.1kb fragments into the pLoxPneo vector.The PCR,restriction enzyme digestion and sequencing analysis results indicated that the anticipated construction was correct.With Lipofectamine 2000,GFF cells were transfected with linear ploxPneo-MSTN,and cultured for twenty-four hours,then added the drugs G418?600?g/mL?and G418?600?g/mL?/GANC?2?M?,respectively.After 12 days of selection,the number of clones was counted and analyzed statistically to confirm the validity of the positive-negative selection vector.5.Goat MSTN gene targeting in GFF cells and identification of positive cell clones.In the cell resistance experiment,non-transfected GFF cells were cultured in DMEM with the drug G418?200?1200?g/mL?.The optimal concentration of G418 was determined to 700?g/mL,in which cells could be killed in seven days.Linear ploxPneo-MSTN vector was transfected into GFF cells with Lipofectamine 2000,for gene targeting to place the Neo gene at the MSTN locus.Transfected cells were cultured in 100-mm dishes for twenty-four hours,then added G418?700?g/mL?to select for twelve days,segregative clones were isolated and expanded in 24-well plate,then added G418?200?g/mL?and GCV?2?M?.247 clones were selected out and could grow,which were tested by PCR and DNA sequence analysis screening for targeting.The results indicated that one cell clone targeting with HR events was obtained.By calculation the absolute efficiency of gene targeting was 7.1×10^-9,and relative efficiency was 4.1×10^-3.