Production Of MSTN Knockout Sheep Via CRISPR/Cas9

Muscle property is one of the important economic characters of animals. The process of muscle growth is influenced by genetic and environmental factors. Studies have shown that many genes involved in the regulation in the process of muscle occurrence and development, among which Myostatin (MSTN) is considered as a key gene related to the body weight and lean meat percentage. MSTN gene, also known as GDF-8 (growth differentiation factor 8), belonging to the TGF beta (transforming growth factor beta, transforming growth factor) super family GDF subfamily. As an important factor negative to regulate muscle growth, MSTN is highly expressed in animal skeletal muscle. Many studies show a negative correlation between its expression level and muscle weight, and the lack of its expression or mutations can cause muscle hypertrophy. Our research combined the CRISPR/Cas9 gene targeting editing technique, monoclonal cell cultivation with somatic cell nuclear transfer technology to product transgenic sheep individuals with MSTN gene deficient expression. Our results can provide reference value for improving the muscle quality and economic benefits of sheep, also have theorial and practical significance for new varieties breeding of meat sheep and enriching biodiversity. The main research contents are as follows:1. Construction and activity verification of MSTN knockout vector:cDNA was obtained from the total RNA of sheep muscle tissue as a template, PCR was performed to amplify the MSTN CDS sequence of, and the purified product was sent for sequencing analysis. No homology target site gRNAs within the whole genome were designed, and digested DNA were constructed comprising gRNAs using in vitro bridge-PCR. Case9 enzyme digestion reaction tested luciferase signal using standard gRNAs with known SSA activity as controls. The sMSTN-Cas9/gRNA vector with high in-vitro activity was built and transfected to SEFs separated from 1-week-old lamb ear tissues using FuGene HD liposome at the ratio of 1:2.5. Positive cells were collected and genomic DNAs were extracted, and a 1000bp fragment was cloned including target sites for T7E1 enzyme activity detection. Sequence analysis showed MSTN was successfully cloned with 100% sheep homology. In vitro activity validation of four gRNA targets showed that enzyme activity were respectively 50%,65%,5%,95%. The gRNA4 target with highest in vitro activity was selected to verify endogenous activity, as a result the mutant genome was successfully cutting to two stripes by T7E1. The results suggested that No.7 target has both exogenous and endogenous active, conforming to the requirements of later experiment.2. Effect of MSTN knockout on the SMSCs differentiation:using two enzymes to separate sSMSCs from skeletal muscle of newborn lamb. After fascia striping, filaments digestion and differential adhesion purification, high purity sSMSCs were obtained in F5 generation. Indirect immunofluorescence detected sSMSCs marker gene Pax-7 and statistical positive cells were found to be 89.3±2.8%, indicating that sSMSCs can be used for subsequent experiments with high purity. Hungrily induction of sSMSCs with 1% serum concentration displayed that a lot of early differentiation of myotubes formed after 72h.After transfecting MSTN-Cas9/gRNA4 vector into sSMSCs and continuously screening by puro, hungrily induction by 72h and detection of myogenic differentiation early marker Desmin protein by indirect immunofluorescence were conducted. Results showed that control group and knockout group myotube formation efficiencies were 11.2±1.3% and 19.5±2.1% respectively, and myotubes average lengths are 22±3.4μm and 47±2.8μm by 1% serum starvation treating 72h, displaying that MSTN knockout can promote satellite cell myogenic differentiation in the number and length.3. Preparation of unmarked MSTN knockout monoclonal cell lines:In order to comply with the tspecifications and requirements of transgenic animal production, restriction sites Mlu I and Spe I located were selected for double digestion reaction to renovate the existing carrier. New vector without marker gene fragments of GFP and puro was transfected to SEFs with 200V voltage.Individual cells was were seeded to 96-well plates with a fine glass needle connecting mouth pipette after 48h. After attaching and propagation, genomic DNA of each monoclonal cell lines were extracted. PCR amplified fragment containing target site using specific primers and products were sent to sequence. Experiments were inoculated four 96-well plates, and cells were observed total in 107 holes, of which 93 holes were mono-cell. Finally,61 monoclonal cell lines were successly amplified, and sequencing results by UCSC alignments showed that three mutant cell lines were obtained with a mutation rate of about 9.3%. No.18 cell line has 21bp target sequence deletion; No.15 cell line generated 29bp mismatches; and No.16 cell line absolutely generated mismatches from front 11bp of target sequence.4. Production of transgenic cloned sheep using SCNT:No.16 cell line was selected as donor cells because of more mutated bases. Sheep mature oocytes with first polar body in vitro separated and matured for 19-22h were selected as receptor cells. Cell nucleuses of receptor were removed and donor cells were injected between the cytomembrane and zona pellucida under microscope, electric shock to make the two fusion. After fusional embryos parthenogenetic activation and development, the equal cleavage embryos were selected surgically implanted into estrus ewes tubal in ovarian ovulation side. Three months later, pregnancy of ewes was detected by B ultrasound. In this experiment, sheep oocytes maturation rate, fusion rate and cleavage rate were 66.0%,69.5%,80.9% up to the national average; 415 embryos were transplant to the fallopian tubes of 20 ewes; B-test results showed that eight ewes became pregnant, and the pregnancy rate was 40%.