Study On Myostatin Gene Knockdown Cloned Sheep

Myostatin(MSTN)is a kind of growth factor,which belongs to the TGF-? superfamily and expressed in skeletal muscle specifically.It plays an important role in negative regulation of muscle cells growth and development.Overexpression of MSTN gene causes muscle atrophy,while artificial knockdown or mutation of MSTN gene can effectively promote the differentiation of myoblasts into mature muscle fibers,increasing the muscle tissue generation,which leads to double-muscular phenomenon.With the rapid development of CRISPR/Cas9 technology,CRISPR/Cas9 technology is widely used in the production of genetically modified animals.In this study,sheep MSTN gene was cloned to construct eukaryotic expression vector,in the meanwhile,MSTN interference sequence was designed and synthesized,and RNA interference vector was constructed;After co-transfected into 293GP cells,RNA interference efficiency was detected by Real-Time PCR and the most efficient siRNA sequence was obtained;After linearized and transfected into sheep fibroblasts,the MSTN knockdown transgenic cell line was screened by G418;Using MSTN knockdown transgenic cells as nuclear donors,MSTN knockdown cloned embryos and transgenic sheep were obtained by nuclear transfer.It is expected that a new method could be established to create a new variety of sheep with massive hyperplasia and hypertrophy of skeletal muscle.In addition,gene editing embryos and mice were obtained by using CRISPR/Cas9 system,which laid the foundation for obtaining gene targeting sheep.Main results and conclusions were showed as follows:1.RNAi expression vector construction and activity analy sis(1)Cloning of sheep Myostatin gene:Referring to Myostatin gene CDS sequence in GenBank(AFO 19622.1),sheep Myostatin gene was cloned by RT-PCR,then it was ligated into pcDNA3.1(+)vector;(2)Construction of RNAi expression vector:Referring to mRNA sequence of sheep Myostatin gene,RNA interference sequence was designed and synthesized and ligated into pRNAT-U6 vector to obtain RNA interference expression vector.After co-transfected into 293 GP cells with eukaryotic expression vector,RNA interference efficiency was detected by Real-Time PCR and the most efficient siRNA sequence was obtained;(3)Expression activity detection of RNA interference vector:After linearized,the RNA interference vectors were injected into pronuclear-stage mouse embryos by using micro injection.Cell cleavage was observed after injection and blastocysts expressing green fluorescent protein were obtained by in vitro culture and the results indicated that the RNA interference vector can be integrated into the mouse genome and expressed nonnally in embryos and support embryonic development.2.Establishment of myostatin gene knockdown fibroblast cell line of sheepLinear RNA interference vectors were transfected into sheep fibroblasts by liposome transfection and transgenic positive cells were obtained after screened by G418.PCR and sequencing results showed that the constructed genes were integrated into the cell genome.The results of morphological characteristics,growth curve and freeze-thaw characteristics of the transgenic positive cells showed that the transgenic cells had normal biological characteristics.3.Generation of MSTN knockdown transgenic embryos(1)Modification and treatment of donor cells:The cleavage rate of nuclear transfer embryos derived from fibroblasts was significantly higher than those of MSTN knockdown transgenic cells(44.6%vs 33.0%),but there was no significant difference in morula/blastocyst rate between the two groups(24.1%and 15.6%respectively).Serum starvation treatment was not necessary for donor cells,contact inhibition could also induce cells into GO phase.(2)Fusion,activation and culture of reconstructed embryos:The fusion efficiency could be significantly improved by using one-by-one fusion method,but there was no significant difference in embryonic development ability between the one-by-one method and batch fusion method;There was no significant difference between A23187+6-DMAP and Ionomycin+6-DMAP in oocyte activation;Culture for 3-4 hours before activation could effectively increase the cleavage and blastocyst rate of embryos;Both KSOM and SOFaa could be used for sheep cloned embryos in vitro culture.4.Embryo transfer and fetal identification of transgenic NT embryos(1)Transgenic NT embryo transfer:242 pronuclear-stage or 2-cell stage embryos were transplanted into 23 recipient sheep with synchronous estrus to test their developmental ability in vivo.(2)Identification of transgenic fetus:Only one of the 23 recipients was pregnant but abortion occurred during pregnancy,failed to obtain a viable individual,and one aborted fetus was obtained;PCR identification of aborted fetus showed that the genome of the fetus was integrated with the targeted gene.5.Generation of muscle-specific gene-targeting embryos expressing Cas9 protein(1)Design and in vitro validation of sgRNA of Rosa26 gene:The sgRNA was designed according to the sequence of Rosa26 gene in mice.In vitro editing experiments showed that the sgRNA designed could edit Rosa26 locus;(2)Construction of muscle-specific targeting vector:The homologous target vector Donor-SP-Cas9-DsRed expressing Cas9 protein specifically in muscle was successfully constructed by PCR and homologous recombination;(3)Generation of Rosa26 gene edited embryos and mice:After injecting Rosa26 sgRNA and Cas9 into mouse embryos,the injected embryos could cleave normally and support blastocyst development.The results of PCR and sequencing showed that the obtained embryos were gene-edited embryos.After embryo transfer,Rosa26 gene-edited mice were obtained;(4)Generation of muscle-specific gene-targeting embryos expressing Cas9 protein:By injecting the homologous targeting vector with Rosa26 sgRNA and Cas9,the muscle-specific gene-targeting embryos expressing Cas9 protein were obtained.In summary,through constructing MSTN gene RNA interference vector,transfecting sheep fibroblasts to establish MSTN knockdown transgenic cell lines and nuclear transfer of transgenic cells,we obtained sheep transgenic cloned embryos with MSTN knockdown,aborted fetal sheep was obtained after embryo transfer which indicated that the reconstructed embryos could develop in vivo and the transgenic sheep with MSTN gene knockdown could be obtained by this method.Our results provide a basis for breeding new sheep strains with MSTN gene knockdown.In addition,gene editing embryos and mice were obtained by using CRISPR/Cas9 system.A gene editing and gene targeting system was initially established in mice,which laid the foundation for obtaining gene targeting sheep.