Studying The Effect Of Follistatin Over-expression On Ovine Myoblast Cells Using Adeno-associated Virus (AAV) In Vitro

Recently, virus-mediated transfer has been employed to generate transgenic animal. Viral vectors provide an alternative, efficient mechanism of delivery. However, there is some controversy regarding the efficiency of transmission of exogenous DNA by viral vectors, such as retroviruses, adenoviruses and lentiviruses. In contrast to retroviruses, recombinant adenovirus-based vectors are able to infect both dividing and non-dividing cells. Adeno-associated viruses are small, nonpathogenic, dependent parvoviruses that can integrate in a site-specific manner into chromosomes. Adeno-associated viral (AAV) vectors transduce a variety of somatic tissues, including skeletal muscle, without eliciting an immune response in mice. These features give the AAV System a superior biosafety rating among gene delivery and expression vectors of viral origin. Moreover, recent reports have indicated that adeno-associated virusesare capable of integrating the FST gene into the host chromosome and facilitating long-term transduction. Follistatin (FST) has been demonstrated to be a potent antagonist of other members of the TGF-β family, including myostatin. Myostatin (MSTN) negatively regulates myoblast differentiation and proliferation. Recent studies have highlighted the potential benefit of inhibiting MSTN, which results in a double muscle phenotype in MSTN-deficient cattle and MSTN-knockout mice. In particular, since sheep are an economically important animal, breeding double muscle sheep is of high economic value. However, AAV-mediated FST gene transfer has not been reported in sheep; whereas there are several reports of FST gene transfer in sheep by other vectors, such as lentiviral vectors. Using of AAV vectors to produce transgenic animal can increase biosafety rating for human. The objective of the current study was to use a recombinant AAV serotype 2 (rAAV2) carrying follistatin to explore the effects of FST on ovine primary myoblast (OPM) differentiation and proliferation in vitro. In the present study, we tested the hypothesis that an AAV-2 virus carrying the FST gene is capable of inducing ovine myoblast differentiation and proliferation in vitro. The main results are summerised as follows:1- The Ujumqin sheep primary myoblast cells had been cultured in vitro. Primary myoblast cultures had been obtained from 50 to 60-day-old sheep fetuses. They had spindle-shaped morphology typical of myoblasts. The growth of myoblasts was very good, and could be used for the next experiments of the plasmid and adeno-associated virus vector transduction.2- The full-length sequence of the gene encoding follistatin, containing a 1035bp segment encoding the 344 amino acid, was cloned vector and sequenced (GenBank Accession No. KF833357). From the follistatin CDS sequence, the amino acid sequence was deduced using software from NCBI. Multiple alignments of the amino acid sequences were produced.3- We had cloned the sheep follistatin gene, and inserted into the eukaryotic expression vector pAAV-IRES-GFP plasmid to construct the sheep follistatin over-expression vector pAAV-CFS-FLAG.4- AAV particles were produced by co-transfection of the recombinant pAAV-CFS-FLAG vector with two helper plasmids (pAAV-RC and pHelper) into 293 cells. The AAV had been transduced into sheep primary myoblasts at the multiplicity of infection (MOI) of 50, and western blot confirmed that AAV2 could successfully express follistatin protein in transduced primary myoblast cells.5- The optical density (at 450 nm) was significantly increased in the transduced primary myoblast cultured in GM media compared with the control, indicating that FST significantly increased proliferation. Moreover, Real-time quantitative PCR results demonstrated that over-expression of follistatin resulted in a dramatic increase in Akt I and CDK2 expression and a decrease in p21 expression. On the other hands, cell cycle analysis confirmed that FST down-regulated p21, a CDK inhibitor, and increased the level of CDK2 expression in OPM cells. Hence, follistatin positively regulated the G1 to S progression. Our findings showed that over-expression of follistatin induced proliferation through a down-regulation of the p21 gene at transcript levels in myoblasts under proliferating conditions.6- Following functional studies and identification of target genes of follistatin, we analyzed whether follistatin expression was regulated by the ERK1/2 pathway in ovine myoblasts. It is widely believed that activation of MAPK pathways, in particular the extracellular signal-regulated kinase (ERK) 1 and 2 (p44 and p42 MAPKs), contributes to cell proliferation. Our results demonstrated that follistatin over-expression substantially induced the ERK activation in these cells. Follistatin over-expresion by AAV2 induced the pERKl and pERK2 levels 1.7 and 2.3-fold (P<0.01), respectively. As expected, ERKs (mainly p42 MAPK) were robustly activated in transduced cells. These results suggest that Follistatin overexpresion induced cell proliferation via the activation of the ERK pathway.7- In exploring the signaling mechanism, we focused on Akt, as it is a well-acknowledged critical signaling node within the cells under both physiological and pathological conditions and it plays a pivotal role in cell survival. Akt was robustly activated in the transduced cells. Our findings indicated that follistatin regulate the ERK1/2 and PI3K/Akt pathway. Our result showed that follistatin induced the phosphorylation of ERK1/2 and Akt, resulting in a significant promotion of ovine myoblast cell proliferation.8- Cultures were stained with Giemsa in order to determine the number of nuclei that had contributed to myotube formation. Fusion percentage was assessed by determining the ratio of the number of myotube nuclei to total nuclei. Fusion percentage in myotubes transduced with AAV-CFS-FLAG was 47.11%, which was significantly greater than the 32.05% observed with non-transduced cells. These findings suggest that the addition of ovine follistatin to differentiation medium resulted in more nuclei contributing to myotube formation than non-transfected myoblasts. Moreover, Real-time quantitative PCR results indicated that follistatin over-expression contributed to an increase of Myogenin, Myo D, Myf5, p57 and p21 mRNA expression and had no effect on myostatin and ActRIIB mRNA expression under differentiating conditions. These findings demonstrate that FST induces differentiation through up-regulation of myogenesis marker genes under differentiating conditions.9- We make use of AAV transgenic approach; the transgenic cells with good growth status were obtained. In the present study, we used flow cytometry technique to sort the transgenic cells. After the first (3 generations) and second (6 generations) sorting, the positive rate of cells was 92.5% and 99%, respectively. The growth status of positive cells was excellent. Southern blot results demonstrated that AAV vectors integrated at apparently random genomic sites and promoted the transgenic myoblast proliferation and differentiation by follistatin over-expression.10- Follistatin over-expression interferes at the G1 phase in transgenic cells. The cell cycle was analyzed using a fluorescence-activated cell sorter (FACS) after staining with propidium iodide. Propidium iodide staining revealed that transgenic cells approximately decreased the G0/G1 population of cells by 12%(P<0.01), suggesting that FST over-expression decreases the G1 arrest. The results confirmed that the increase in myoblast proliferation was due to a decrease in the number of cells in G1 phase. Moreover, these findings demonstrated that AAV-FST over-expression decreased number of cells in G1-phase in transgenic clone, and therefore made the transition to the S (DNA synthesis)-phase.These results expanded our understanding of the regulation mechanism of FST in ovine primary myoblasts. Our results provide the first evidence that the AAV viral system can be used for gene transfer in ovine myoblast cells. Moreover, the results showed that an AAV vector can successfully induce the expression of FST in OPM cells in vitro.