40 results about "Myostatin Gene" patented technology

This invention relates to compounds, compositions, and methods useful for modulating myostatin (GDF8) gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of myostatin gene expression and / or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of myostatin genes.

The invention provides a method for knocking off an animal myostatin gene by using a CRISPR-Cas9 system. The method comprises the following steps: firstly, acquiring a DNA sequence aiming at an sgRNA recognition area of a second myostatin exon, wherein the base sequence of the DNA sequence is as shown in SEQ ID NO.1; secondly, establishing an sgRNA expression structure of the second myostatin exon, inserting a T7 starter before an sgRNA transcriptional start site, establishing an in-vitro transcription carrier of Cas9 protein, and regulating and controlling by using the T7 starter. Cas9 mRNA and sgRNA are obtained through the in-vitro transcription carrier of Cas9 and sgRNA, and the method can be used for knocking off the animal myostatin gene.

This invention relates to compounds, compositions, and methods useful for modulating myostatin (GDF8) gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of myostatin gene expression and / or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of myostatin genes.

The invention discloses a porcine myostatin gene editing site and application thereof, and belongs to the fields of biotechnology and biomedical medicine engineering. A gene editing target site is separated from a porcine myostatin gene editing area, the sequence of the gene editing target site is 5'-GGCTGTGTAATGCATGTATGTGG-3', and the gene editing target site is located at a first exon of an MSTN (myostatin) coding area. The site can be recognized by Cas9 endonuclease specifically, so that double strands are medicated to break, homologous recombination between the double strands and shooting carriers is achieved, and mutant genes or selectable maker genes are integrated to a pre-positioned site of a recipient cell genome. A statistical result shows that shooting efficiency of the site is 80.5%. The porcine myostatin gene editing site has the advantages that an effective drone is provided to accurate porcine myostatin gene editing, a new strategy is provided to development of new species of live pigs with high lean meat percentage, and reliable measures and materials are provided to study on molecular mechanism and signal channel of myostatin.

The invention discloses a polypeptide pair for specifically recognizing a muscle myostatin gene as well as an encoding gene and an application of the gene. The polypeptide pair provided by the invention comprises a polypeptide I and a polypeptide II; double-chain amino acid in the polypeptide I sequentially comprises 2-3, 36-37, 70-71, 104-105, 138-139, 172-173, 206-207, 240-241, 274-275, 308-309, 342-343, 376-377, 410-411, 444-445, 478-479 and 512-513 of the sequence 3; and double-chain amino acid in the polypeptide II sequentially comprises 2-3, 36-37, 70-71, 104-105, 138-139, 172-173, 206-207, 240-241, 274-275, 308-309, 342-343, 376-377, 410-411, 444-445, 478-479, 512-513 and 546-547 of the sequence 5. The polypeptide pair provided by the invention can specifically recognize the muscle myostatin gene, and has important application value in obtaining high-quality pig breeds with high lean meat percentage by knockout or modification of MSTN on pigs.

This invention relates to compounds, compositions, and methods useful for modulating myostatin (GDF8) gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of myostatin gene expression and / or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of myostatin genes.

The present invention provides methods comprising the in vivo delivery of small nucleic acid molecules capable of mediating RNA interference and reducing the expression of myostatin, wherein the small nucleic acid molecules are introduced to a subject by systemic administration. Specifically, the invention relates to methods comprising the in vivo delivery of short interfering nucleic acid (siNA) molecules that target a myostatin gene expressed by a subject, wherein the siNA molecule is conjugated to a lipophilic moiety, such as cholesterol. The myostatin siNA conjugates that are delivered as per the methods disclosed are useful to modulate the in vivo expression of myostatin, increase muscle mass and / or enhance muscle performance. Use of the disclosed methods is further indicated for treating musculoskeletal diseases or disorders and / or diseases or disorders that result in conditions in which muscle is adversely affected.

The invention discloses a molecular marker for identifying duck slaughter traits based on a myostatin gene MSTN as well as an identification method and application of the molecular marker. The molecular marker is T or C, the molecular marker is located at the 458th site of a myostatin gene MSTN, and the myostatin gene MSTN has a nucleotide sequence as shown in SEQ ID NO. 1. The duck slaughter traits are selected according to genotypes by utilizing the polymorphism of the 458th basic group on the CDS sequence exon1 of the MSTN gene. The method for selecting the slaughter traits of the ducks has the advantages of early selection, time saving, simple operation, cost saving, high accuracy and the like.

The invention provides a method for knocking out a bovine myostatin gene by using zinc finger nuclease. The method comprises the following steps of: designing a ZFNs specific site expression vector according to a bovine myostatin gene sequence and transferring into bovine fibroblasts to obtain cells in which the myostatin gene is knocked out. The one-time transfection can be realized by utilizingZFNs-mediated gene knockout so as to obtain cell clone in which a diallele is knocked out, which is difficult to realize in the conventional gene targeting process; therefore, the medicine screening process is saved, the formation of cell monoclone is facilitated, the process that cells are required to resist the poisoning of medicines is avoided, key effects are achieved on the subsequent somatic cell nuclear transplantation and the development quality of embryos, resistance genes are not contained at the same time, and the biological safety evaluation process is greatly simplified.

The invention discloses a constructing method of a goat Myostatin gene knockout carrier. In the method, according to sheep Myostatin gene sequence, two pairs of PCR (Polymerase Chain Reaction) primers are designed, genome DNA (Deoxyribonucleic Acid) extracted from goat fetus fibroblast is used as a template, amplification is carried out by adopting high-fidelity long-fragment Taq enzyme and a Long-PCR method to obtain homologous arms, wherein the homologous long and short arms are 4.6kb and 1.9kb, respectively. In order to verify whether the sequence of the obtained homologous arms can be used or not, the amplified fragments are respectively cloned to a pMD18-T carrier, and sequencing and homology comparison are carried out after enzyme digestion and PCR identification; and then the homologous long and short arms are respectively cloned to a carrier pLOXP and then identified by enzyme digestion and RCR method, and screening is carried out to obtain a MNST gene targeting carrier pLOXP-MSTN containing neo and HSV-tk positive-negative screening marker genes. According to the invention, the Myostatin gene knockout carrier is constructed by the Long-PCR method, and goat fetus fibroblast is transfected to obtain gene knockout cells; and by adopting the technique, animals with high lean meat percentage can be obtained, and a MNST gene knockout goat model is constructed, so that the method has important significance in further researching the function of the gene in goat.

The invention discloses a method for deleting sheep Myostatin gene locus by zinc finger nuclease, and the method comprises the following steps: designing a sequence which targets a sheep Myostatin gene exon 3 and can recognize a specific target site of 39bp, constructing a zinc finger nuclease plasmid, transfecting a sheep fibroblast by the plasmid, obtaining a monoclonal cell with deleted diallele with 95bp and 113bp. The invention demonstrates that the method can accurately delete Myostatin gene exon 3 and separate cells whose Myostatin gene locus is deleted, thereby confirming the effectiveness of the sequence, and the obtained monoclonal cell with deletion is not need to be screened by antibiotic. The invention has two advantages of genome targeting modification and safety, and has important meanings for cultivating transgenic sheep without resistance marker genes and with safety and high efficiency.

A pig myostatin gene promoter and its applications are disclosed in the present invention. The DNA fragment provided in the present invention which is derived from pigs is anyone of the following DNA molecules 1)-4): 1) the DNA molecule of SEQ ID NO:2 shown in the Sequence Listing; 2) the DNA molecule of SEQ ID NO:3 shown in the Sequence Listing; 3) the DNA molecule which hybridizes to the DNA sequence in 1) or 2) under strict conditions and has promoter function; 4) the DNA molecule which has more than 90% homology with the DNA sequence in 1) or 2) and has promoter function. The experiments in the present invention prove that the promoter provided in the present invention can drive the expression of firefly luciferase reporter gene. The promoter can also be built into the reporter vector and then the vector is transfected into cultured swine and human cells, and the activity and efficiency of the promoter are identified by accurate quantitative methods through reporter gene test.

The invention belongs to the field of fish genetics and breeding, and specifically discloses a method for efficiently creating mstn gene mutants of Yellow River catfish. This method is aimed at the Yellow River catfish Myostatin‑ Myostatin Gene Design Target Site, Synthetic gRNA, and Cas9 Proteins were co-microinjected into mature eggs of Yellow River catfish (stage V eggs), and then dry insemination was performed with yellow catfish sperm. After hatching and emergence, the mutation efficiency was tested. The knockout efficiency was 80%, and the average mutation efficiency was 74.1%. And the proportion of fully mutant individuals was 24%. Compared with the traditional fish gene editing method, this method not only obtains higher mutation efficiency, but also can efficiently obtain complete mutant individuals of Yellow River catfish, and the combination of gynogenesis increases the homozygosity of mutant individuals. For economic fish with a long reproductive cycle The rapid directional molecular design breeding of species also has important reference value.

The invention discloses a curative vaccine for Myostatin specific antibody and the method for preparing the same. The human myostatin C-end gene section that is connected with Th cell auxiliary epitope TT830-844 at N-end is prepared by using artificial DNA synthesizing method. The genetic sequence is TT-Ms, and the TT-Ms genetic section is cloned to pQE30 pronucleus expression carrier to constructpQE-TT-Ms recombinant expression carrier, and constructs pQE-Ms recombinant expression carrier by PCR augmenting Ms genetic section. The fused protein His-TT-Ms and His-Ms is expressed in bacillus coli, and is used as protein vaccine for myostatin after certification. The synthesized TT-Ms genetic section is cloned to specific plasmid of pVAC1-cms gene vaccine, and constructs pVAC-TT-Ms recombinant plasmid, and it is used as myostatin gene vaccine after external certification for target protein expression. The constructed Myostatin protein vaccine and gene vaccine is used in healthy adult BALB / c mouse, the specific antibody of myostatin molecule for human can be induced, and the quality and force of skeletal muscles of mouse is obviously improved.

The invention discloses a polypeptide pair for specifically recognizing a muscle myostatin gene as well as an encoding gene and an application of the gene. The polypeptide pair provided by the invention comprises a polypeptide I and a polypeptide II; double-chain amino acid in the polypeptide I sequentially comprises 2-3, 36-37, 70-71, 104-105, 138-139, 172-173, 206-207, 240-241, 274-275, 308-309, 342-343, 376-377, 410-411, 444-445, 478-479 and 512-513 of the sequence 3; and double-chain amino acid in the polypeptide II sequentially comprises 2-3, 36-37, 70-71, 104-105, 138-139, 172-173, 206-207, 240-241, 274-275, 308-309, 342-343, 376-377, 410-411, 444-445, 478-479, 512-513 and 546-547 of the sequence 5. The polypeptide pair provided by the invention can specifically recognize the muscle myostatin gene, and has important application value in obtaining high-quality pig breeds with high lean meat percentage by knockout or modification of MSTN on pigs.

The invention discloses a skeletal muscle specific miRNA expression vector and a reconstitution cell of target Myostatin gene. Transgenic cloned embryos are prepared by the following steps: firstly constructing a skeletal muscle specific miRNA expression vector pmiR-MNM2 containing pre-miRNA and MYL1 gene promoters of the target Myostatin gene; then guiding the exogenous expression vector pmiR-MNM2 into red Augus neonatal bovine fibroblasts by an electric dye-transfer method, obtaining positive cells through G418 screening, and performing PCR (Polymerase Chain Reaction) identification to verify that the target gene is integrated into the genome of bovine embryonic fibroblasts; transferring bovine embryonic fibroblasts subjected to pmiR-MNM2 dye transfer, which are taken as donor cells, into bovine enucleated oocytes to finally obtain the transgenic cloned embryos which are transplanted into wombs of acceptor cows. The invention lays a solid foundation for production of red Augus cloned cattle with a dual-muscle characteristic.

The invention discloses a siRNA capable of restraining chicken myostatin (MSTN) gene expression and application of the siRNA; small interfering RNA (ribonucleic acid) acted on myostatin gene is provided by the invention and is a) or b) or c) as follows: a) double-stranded RNA composed of a sequence 1 of a sequence table and a sequence 2 of the sequence table; b) double-stranded RNA composed of a sequence 3 and a sequence 4 of the sequence table; and c) double-stranded RNA composed of a sequence 5 and a sequence 6 of the sequence table; according to the invention, the small interfering RNA effectively capable of restraining chicken MSTN gene expression is obtained and is proved to effectively reduce expression of MSTN through real-time fluorescent quantitative PCR (polymerase chain reaction) on molecular biology; chicks with obviously enlarged skeletal muscle is successfully acquired through a micro-injection test on embryology; above-mentioned results show that the small interfering RNA provided by the invention can be used for breeding the chicks, so that chicken yield of the chicks specifically high-quality chicks is increased.

The invention relates to a method for modifying an animal genome by directed mutagenesis, in particular relates to a novel directional transformation method of a specific gene of an animal genome by utilizing zinc finger nuclease. The method comprises the following steps: designing the zinc finger nuclease which specifically identifies and cuts a targeting gene according to a sequence in a target animal target gene region; validating the directional cutting capability and efficiency of the zinc finger nuclease to the target gene by using an independent expression system; and carrying out directional genetic modification on the target gene of the embryo of a target animal to obtain a stable genetic character by utilizing the selected zinc finger nuclease. Furthermore, the invention also relates to a main gene mutation mode which is obtained by using the independent expression system, selecting and utilizing the zinc finger nuclease, and a filial generation obtained through efficient screening and oriented genetic modification by utilizing the selected gene mutation mode. On the other hand, the invention relates to the zinc finger nuclease which is obtained by utilizing the method and is capable of specifically identifying the myostatin gene of pelteobagrus fulvidraco and directionally knocking out the myostatin gene of the pelteobagrus fulvidraco.

The invention discloses a transformant, which comprises a recipient bacterium and a recombinant vector transformed into the recipient bacterium, wherein the recipient bacterium is Lactococcus lactis NZ9800; an original vector of the recombinant vector is pNZ8112; and a foreign target gene of the recombinant vector comprises a chicken myostatin gene, a cholera toxin B subunit gene and a cell wall anchor element M6 gene. The transformant can enter the intestinal tract of chicken in an oral administration mode, and has the advantages of reducing the toxicity caused by directly contacting cyclic system and improving the safety of vaccine compared with injection immunity. An antigen gene / immune-enhancing factor is selected for combined immunity, so the immune response of organisms is enhanced and the immune effect is improved.