45 results about "Myosatellite cell" patented technology

The invention employs a CRISPR-Cas9 system to complete mediation of a goat Tbeta4 gene for site-directed knock-in. A gRNA expression vector and a Tbeta 4 homologous recombinant vector based on the CRISPR-Cas9 system are constructed according to a CCR5 gene sequence of the goat. An optimized CRISPR-Cas9 vector and a constructed gRNA expression vector and the Tbeta 4 homologous recombinant vector are transferred to skeletal muscle satellite cells together, in order to obtain cells with the site-directed Tbeta4 gene. A targeting vector constructed based on the CRISPR-Cas9 system provides a simple, fast and safe pathway for site-directed knock-in of the goat Tbeta4 gene. Any screening marker genes are not related in a cell line screening process, safety of transgenic animals is greatly improved, and the method has important value for genetic breeding of goats and research of gene function.

The invention discloses an in-vitro culturing method for a porcine skeletal muscle satellite cell (SMSC). The method comprises the following steps of: collecting a sample; pretreating a culture dish; transplanting skeletal muscle fibers; removing single muscle fibers; subculturing the SMSC; identifying the SMSC; and detecting the purity of the SMSC. A simple and practical high-purity porcine SMSC in-vitro culturing method is established, so that materials are provided for the researches of gene transfer of SMSC mediation, muscle tissue regeneration and developmental mechanism, and a basis is laid for the treatment of muscle-related diseases through skeletal muscle single fibers or SMSC transplanting.

Methods for in vitro isolation and culture and induced differentiation of bovine muscle satellite cells relate to the methods for in vitro isolation and culture and induced differentiation of cells. The invention solves the following problems: the cost is high and the heredity stability is not good in the existing method for isolation and culture of bovine muscle satellite cells, and the obtained myotubes are fewer and do not have contracting function in the existing method for induced differentiation of bovine muscle satellite cells. The method for in vitro isolation and culture of bovine muscle satellite cells is characterized by cleaning the tissues and digesting the tissues with trypsin and collagenase solution; carrying out centrifuging after filtering, re-suspending the cells and culturing the cells by a differential velocity adherent method after inoculation; and culturing the cells until the merging rate is 95% and carrying out heredity with pancreatin. The method for induced differentiation of bovine muscle satellite cells is characterized by obtaining PEI-plasmid complexes; culturing the bovine muscle satellite cells until the merging rate is 75%, adding high-glucose DMEM culture solution after cleaning, adding the PEI-plasmid complexes to the surfaces of the cells, changing culture solution A for culturing and then changing culture solution B for induced differentiation. In the method, the cost for isolation and culture of the bovine muscle satellite cells is low, the heredity stability is good and the myotubes obtained through induced differentiation are large in quantity and have contracting function.

The invention relates to a marine fish cell culture technology, in particular to an aralichthys olivaceus muscle satellite cell line establishing method, a specific primer for identifying a paralichthys olivaceus muscle satellite cell marker gene and application of the specific primer. The aralichthys olivaceus muscle satellite cell line establishing method comprises the following steps: performing primary culture and subculture of aralichthys olivaceus muscular tissues, and then establishing a cell line; after establishing, performing freezing preservation, resuscitation and identification, wherein a cell culture fluid adopted to primary culture and subculture is obtained through adding fetal calf serum and a human basic fibroblast growth factor into the cell culture fluid. The built aralichthys olivaceus muscle satellite cell line is in the format of fusiform or spindle-shaped monocytes, allows continuous passage, can provide a great number of aralichthys olivaceus muscle satellite cells, and not only can be directly used for researches on aralichthys olivaceus muscle development related functional genes to provide rich cell sources for researches on the molecular mechanism of a fish muscle differentiation pathway, but also is expected to provide a research platform for researches and application of muscle character improvement during aralichthys olivaceus culture production.

The invention relates to a method for promoting myogenic differentiation of bovine skeletal muscle satellite cells. According to the method, myogenic differentiation of bovine skeletal muscle satellite cells is promoted by interfering lncRNA-HZ5 expression. The specific method comprises the steps as follows: siRNA of lncRNA-HZ5 is designed and synthesized according to an lncRNA-HZ5 base sequence, siRNA is used for transfection of the bovine skeletal muscle satellite cells, the expression level of lncRNA-HZ5 is lowered, and myogenic differentiation of the bovine skeletal muscle satellite cells is promoted, wherein the genome base sequence of lncRNA-HZ5 is shown in SQ1. The myogenic differentiation process of the bovine skeletal muscle satellite cells regulated by changing lncRNA-HZ5, enlightenment is provided for long non-coding RNA research of muscle development differentiation, and a new idea and reference are provided for clinical research, diagnosis and treatment of muscle development differentiation and damage repair.

The invention relates to a method for facilitating in vitro multiplication of a bovine skeletal muscle satellite cell. The method comprises the following steps: obtaining a target sequence containing a bovine miR-133b gene sequence; building an expression vector pEGFP-C1-miR133b; transfecting the bovine skeletal muscle satellite cell by the pEGFP-C1-miR133b. By application of the method for facilitating in vitro multiplication of the bovine skeletal muscle satellite cell, the in vitro multiplication ability of the bovine skeletal muscle satellite cell is improved by obtaining over-expression miniature RNA miR-133b of the bovine skeletal muscle satellite cell and using the effect of the miR-133b, and differentiation is effectively inhibited. The over-expression vector of the miR-133b is built by using the pEGFP-C1, and amplification and differentiation of a bovine muscle satellite cell are regulated by the miniature RNA, so as to obtain a high-purity bovine muscle satellite cell. A good cell model is provided for researching the function and the effect of each regulatory factor in amplification and differentiation processes of the muscle satellite cell.

The invention discloses a separation and purification method for skeletal muscle satellite cells in goats. The separation and purification method comprises separation of the skeletal muscle satellite cells in the goats and purification of the skeletal muscle satellite cells in the goats. According to the method, trypsin digestion and a tissue mass method are combined for separation of the skeletal muscle satellite cells, a differential adhesion method is used continuously for purification of the skeletal muscle satellite cells in the goats during cell passage and is used for purification of the skeletal muscle satellite cells in the goats during more than 5 successional generations in late passage process, and the skeletal muscle satellite cells with higher purity are obtained.

The invention belongs to the field of biotechnology, and especially relates to an in vitro culturing method of goat skeletal muscle satellite cells. The in vitro culturing method comprises following steps: (1) sampling; (2) digestion; (3) filtration; (4) primary culturing; and (5) induction culturing. According to the in vitro culturing method, goat skeletal muscle satellite cells can be quickly obtained via in vitro culturing, and are uniform in cellular morphology, and high in breeding vitality; and after differentiation induction, myotubes can be obtained via fusion of the goat skeletal muscle satellite cells. The in vitro culturing method of goat skeletal muscle satellite cells is simple in operation, is capable of reducing culturing cost greatly, possesses certain economic significance, and can be used for obtaining a large amount of cells with excellent subculturing stability. In vitro culturing of goat skeletal muscle satellite cells is capable of providing exploration on molecular mechanisms of muscle differentiation approaches and production of transgenic animals with improved muscle properties with abundant cell resources, and possesses theoretical values and application values in a certain degree.

The invention provides a simple extraction method of high-purity mouse skeletal muscle satellite cells, which is characterized by comprising the concrete steps that: step 1, bupivacaine hydrochloride is injected at the limb muscle of a mouse; step 2, the mouse is killed, the limb muscle is winkled, vessels, fat and fascias are removed, the muscle is sheared into small granules being 0.5 to 1.5mm<3>; step 3, mixed digestive enzyme is added for incubation, the cell suspension liquid is filtered, the obtained filter liquid is centrifuged, the supernate is abandoned, and a growth culture medium for cell precipitation is suspended again; step 4, 60 percent percoll separation liquid, 20 percent percoll separation liquid and cell suspension liquid are respectively injected along the tube wall, a long-head suction tube is used for sucking the cell suspension liquid at the interface of the 20 percent percoll separation liquid and the 60 percent percoll separation liquid, and cell precipitates are obtained through centrifugation; and step 5, the cell precipitates are cultured, and the mouse skeletal muscle satellite cells are obtained. The method is simple, easy and fast, the purification rate and the yield of the skeletal muscle satellite cells are high, higher multiplication and differentiation capability is realized, and the external gene modification or the internal transplantation can be carried out.

The invention discloses an application of non-coding small RNA (ribonucleic acid) miR-192 in inhibition of myogenic differentiation of sheep skeletal muscle satellite cells and promotion of proliferation of the sheep skeletal muscle satellite cells. Analogues of the non-coding small RNA miR-192 are transfected to the sheep skeletal muscle satellite cells to result in overexpression of miR-192 in the cells, the sheep skeletal muscle satellite cells with miR-192 overexpression are compared with cells with control nucleotide sequence overexpression, and the differentiation of the sheep skeletal muscle satellite cells is inhibited while the proliferation capacity is improved. The invention further provides an application of the miR-192 in inhibition of RB1 protein level expression in the sheep skeletal muscle satellite cells. According to the invention, functions of the miR-192 for negatively regulating and controlling the myogenic differentiation of the sheep skeletal muscle satellite cells and promoting the proliferation of the sheep skeletal muscle satellite cells are discovered for the first time.

The invention relates to a method for separating and purifying high-purity chicken-precursor intramuscular fat cells and establishing an intramuscular-fat-cell-and-muscle-satellite-cell co-culture system. The method includes the steps that chicken breast muscle tissue is cut into meat paste, and the meat paste is digested with I-type collagenase and centrifuged; upper-layer mature intramuscular fat cells are sucked, inoculated into a culture bottle and subjected to dedifferentiation treatment, and the chicken-precursor intramuscular fat cells are finally obtained; lower-layer cells obtained after centrifuging are precipitated and resuspended, the cells are purified in a differential-wall-attachment mode, and high-purity muscle satellite cells are obtained; the precursor intramuscular fat cells and the muscle satellite cells are respectively inoculated into a transwell chamber or a matched culture plate, and the co-culturing system is established. By means of the method, the technical bottleneck that the chicken-precursor intramuscular fat cells cannot be independently separated and purified all the time is broken through, and the method can be applied to quantitatively simulating muscular tissue, accurately researching the interactional relationship and the molecular regulation mechanism of the chicken intramuscular fat cells and muscle cells in vitro, and screening, researching and developing related nutrients or medicine.

The invention provides a lncRNA (long noncoding RNA) encoding gene sheep related to skeletal muscle development as well as application thereof. The lncRNA related to different development stages of Dorang sheep skeletal muscle is identified and analyzed by transcriptome sequencing, the function of the lncRNA is research in the cell level, and the lncRNA which obviously influences the skeletal muscle development is screened. The invention firstly discovers that overexpression of lncRNA TCONS 00544451 has the functions of inhibiting skeletal muscle satellite cell proliferation and promoting cellmyogenic differentiation, and scientific basis and technical support are provided for molecular breeding and variety improvement for improving sheep meat producing performance.

The invention belongs to the technical field of animal cell culture, and particularly relates to a porcine skeletal muscle satellite cell culture medium PM+ and application thereof. Every 1L of the orcine skeletal muscle satellite cell culture medium PM+ comprises 20% of fetal calf serum (FBS), 1% of cell culture additive (GlutaMax), 1% of non-essential amino acid (NEAA), 1% of antibiotic-antifungal (AA), 0.5% of chick embryo cultyre, 50 mg / L gentamycin, 25 mu L / L alkaline fibroblast growth factor, and the balance of RPMI 1640 liquid basal culture medium. The invention also discloses application of the culture medium.

The invention relates to a marine fish embryo cell culture technology, in particular to an establishing method of a myosatellite cell system of bastard halibut during an embryonic period. The establishing method comprises the following steps: carrying out in-vitro separation on an embryo during a bastard halibut kirschner capsule formation period, removing an egg membrane, dispersing cells, and carrying out primary culture and secondary culture, thus establishing the myosatellite cell system during the embryonic period. The form of the myosatellite cell system, established by the invention, ofthe bastard halibut is fusiform or spindle-shaped mononuclear cells; myofiber can be differentiated to form by culturing for 5 days or above within a single generation or carrying out induction of anexogenous factor (horse serum). The myosatellite cell system is capable of supplying a large amount of myosatellite cells, and GFD (Green Fluorescent Protein) transfection and cynoglossus semilaevisspleen and kidney necrosis virus challenge assay detection verify that the myosatellite cells can be normally transfected or infected, so that the myosatellite cell system not only can be directly used for researching functional genes related to muscle growth and development of fishes and providing research materials for exploring a molecular mechanism for induction, proliferation and differentiation of muscle cells of the fishes, but also is capable of providing a platform for improved research and application of muscle characters in bastard halibut breeding.

The invention relates to a method for promoting myogenic differentiation of bovine skeletal muscle satellite cells. The method is characterized in that according to the method, the myogenic differentiation of bovine skeletal muscle satellite cells is promoted by increasing the expression of lncRNA-23, wherein the genome base sequence of the lncRNA-23 is SEQ NO.1. The method can provide inspirationfor researching long non-coding RNA of muscle development differentiation, can lay a good foundation for further researching the action mechanism of key lncRNA in the muscle development process, andprovides inspiration for exploring the muscle growth and development mechanism and improving the yield and quality of beef.

The invention relates to an in-vitro separation culture technique for the skeleton satellite cells of excellent ice and snow athletes, and belongs to the interdiscipline field of cytobiology, sports traumatology and tissue engineering. The technique comprises the following steps: cleaning muscular tissues by using 0.9% sterile saline solution; digesting and separating the skeleton satellite cells under the cooperative action of 1% of I-type collagenase and 0.1% of pronase under the condition of 37 degrees; culturing the skeleton satellite cells by using a high-glucose DMEM (Dulbeccos Modified Eagles Medium) culture medium containing 10% of fetal calf serum; and freezing and storing the skeleton satellite cells by using a freezing-stored liquid formed by the DMEM basal culture medium, dimethyl sulfoxide and the fetal calf serum which are in the ratio of 5:1:4. Thus, the simple, efficient and stably-inherited in-vitro separation cultivation method for the skeleton satellite cells of the excellent ice and snow athletes is provided.

The invention discloses a bovin CMYA1 muscle specific expression core promoter and a preparation method thereof. The preparation method comprises the following steps: firstly cloning a full-length fragment 1155bp of the bovin CMYA1 muscle specific expression core promoter, building a pEGFP-1-CMYA1 promoter carrier, and transfecting mice C2C12 myoblast, bovine skeletal muscle satellite cells and bovine fibroblast to prove the muscle specificity expression of the carrier; then, carrying out the deficiency research on CMYA1 promoter, totally designing 8 deletion fragments of the CMYA1 promoter, respectively building deletion fragment carriers of pGL3-basic-CMYA1 promoters, and transfecting C2C12 cells; and finally verifying, thereby obtaining the CMYA1 core promoter of which the gene sequence is SQ2. The CMYA1 promoter is specifically expressed in muscle cells, and the promotion activity is high; moreover, such the characteristic can be utilized by people to make a research on the development and differentiation of muscles.

The invention discloses an application of a ferroptosis inhibitor in preparation of a preparation for improving the motion function of aged individuals. Through transcriptome sequencing analysis, Tfr1 expression in skeletal muscle and muscle satellite cells is reduced along with age increase. The specific knockout of the Tfr1 muscle satellite cells leads to irreversible inactivation of the muscle satellite cells. The skeletal muscle regeneration process of the Tfr1 muscle satellite cell specific mouse is accompanied by amyotrophy, iron ion accumulation and increase of unsaturated fatty acid biosynthesis, and then skeletal muscle ferroptosis is induced. The senescent skeletal muscle shows that the expression of Tfr1 is reduced, and the surface of an Slc39a14 membrane is enriched, so that the absorption of non-iron transporter binding iron is promoted to be increased, and the occurrence of ferroptosis is promoted. By intraperitoneal injection of the Ferrostatin-1 into aged mice, muscle aplastic disorder and dyskinesia caused by skeletal muscle ferroptosis can be remarkably improved.

The invention discloses a method for inhibiting proliferation and myogenic differentiation of bovine skeletal muscle satellite cells by interfering UBA2 expression, the method realizes inhibition of proliferation and myogenic differentiation of bovine skeletal muscle satellite cells by interfering UBA2 expression, and the genome base sequence of UBA2 is SEQ NO.1. According to the method, the proliferation and myogenic differentiation process of the bovine muscle satellite cells is regulated and controlled by changing UBA2 expression, enlightenment can be provided for research of ubiquitin-like modifier activating enzyme for muscle development and differentiation, and a new thought and reference are provided for clinical research and diagnosis and treatment of muscle development and differentiation and damage repair.

The invention relates to a method for interfering real-time fluorescent quantitative PCR reference gene screening of MSTN bovine skeletal muscle satellite cells. The method comprises the following steps of culturing wild type and transfecting interfering MSTN and contrast bovine skeletal muscle satellite cells, extracting RNA, and carrying out reverse transcription; selecting a plurality of candidate reference genes, carrying out fluorescent quantitative PCR reaction, and making a standard curve; judging the amplification efficiency of primers and the linear relationship between the template concentration and a Ct value; and analyzing the expression stability of the candidate reference genes, and taking the candidate reference gene with the optimal expression stability as the screened reference gene. According to the method, a proper reference gene is researched and screened out for interfering gene expression of the MSTN bovine skeletal muscle satellite cells, a powerful guarantee isprovided for accuracy of fluorescent quantitative PCR detection for interfering gene expression of the MSTN bovine skeletal muscle satellite cells, and reference can be provided for gene expression analysis of the bovine skeletal muscle satellite cells in different treatment and different periods in the future.

The invention relates to an in-situ hybridization probe primer, a probe and a mapping method of the muscle satellite cell of sebastes schlegeli and belongs to the field of molecular biology. A probe template has a primer sequence as shown in SEQ. NO 1 and SEQ. NO 2 and has a probe sequence as shown in SEQ. NO 3. The invention further provides a method for performing in-situ hybridization mapping on the muscle satellite cell of sebastes schlegeli by utilizing the probe. According to the method, the muscle satellite cell of sebastes schlegeli is mapped through skeletal muscle tissue in-situ hybridization of a Pax7 gene, theoretical and technical support can be provided to molecular mechanism study for exploring updating and activating of the muscle satellite cell, and further study on a muscle growth differentiation rule of sebastes schlegeli is favored. Furthermore, the method has a certain application prospect in the aspects of gene mapping and functional study on muscle tissue in-situhybridization of other fishes.

The invention relates to an in situ hybridization probe primer, probe and positioning method of Xu's flat scorpion muscle satellite cells, which belong to the field of molecular biology. The primer sequences of the probe template are as SEQ.NO 1 and SEQ.NO 2, and the probe The sequence is shown in SEQ.NO 3. The invention also provides a method for using the probe to carry out in situ hybridization to localize the satellite cells of the flat scorpionfish muscle. The invention locates Xu's flat scorpion muscle satellite cells through the in situ hybridization of skeletal muscle tissue of Pax7 gene, provides theoretical and technical support for exploring the renewal and activation molecular mechanism of muscle satellite cells, and will help to further study Xu's flat Muscle growth and differentiation rules of scorpionfish; in addition, the invention has a certain application prospect in the research of gene positioning and function by in situ hybridization of muscle tissues of other fishes.

The invention provides a lncRNA (long noncoding RNA) encoding gene sheep related to skeletal muscle development as well as application thereof. The lncRNA related to different development stages of Dorang sheep skeletal muscle is identified and analyzed by transcriptome sequencing, the function of the lncRNA is research in the cell level, and the lncRNA which obviously influences the skeletal muscle development is screened. The invention firstly discovers that overexpression of lncRNA TCONS 00544451 has the functions of inhibiting skeletal muscle satellite cell proliferation and promoting cellmyogenic differentiation, and scientific basis and technical support are provided for molecular breeding and variety improvement for improving sheep meat producing performance.

The invention relates to the application of ubiquitin-specific protease 8 (USP8) in regulating the meat quality and breeding of Hu mutton. Experiments have found that USP8 can affect the eating quality of Hu sheep muscle. The higher the expression level of USP8, the better the eating quality of Hu sheep. Therefore, Hu sheep with good meat quality can be selected as breeding sheep by screening the expression level of USP8. The invention clarifies that USP8 has an inhibitory effect on the early differentiation process of muscle satellite cells, provides a basic theoretical basis for the further protection and breeding of Hu sheep breeds, and also provides some references and technical reserves for the future directional selection of new strains .

The invention discloses a promoter of a bovine alpha-actin gene and an application thereof, belonging to the technical field of genetic engineering. The promoter is characterized in that the nucleic acid sequence of 170bp containing three SRE positive regulation elements inside the 262bp promoter segment of the bovine alpha-actin gene is subjected to PCR amplification by use of different primers to obtain two nucleic acid sequences of 170bp, and the two nucleic acid sequences of 170bp are connected in series with the upstream of the 262bp promoter segment (namely, 170bp-170bp-262bp) to build a promoter segment of alpha-actin, wherein the nucleotide sequence is shown by Seq ID No:1. The promoter has relatively high promoting activity and skeletal muscle specificity and can remarkably improve the expression activity of the alpha-actin gene in bovine skeletal muscle satellite cell and can be used as an efficient and specific skeletal muscle promoter and applied to the transgenic beef production for increasing the muscle output.

The invention relates to a method for facilitating in vitro multiplication of a bovine skeletal muscle satellite cell. The method comprises the following steps: obtaining a target sequence containing a bovine miR-133b gene sequence; building an expression vector pEGFP-C1-miR133b; transfecting the bovine skeletal muscle satellite cell by the pEGFP-C1-miR133b. By application of the method for facilitating in vitro multiplication of the bovine skeletal muscle satellite cell, the in vitro multiplication ability of the bovine skeletal muscle satellite cell is improved by obtaining over-expression miniature RNA miR-133b of the bovine skeletal muscle satellite cell and using the effect of the miR-133b, and differentiation is effectively inhibited. The over-expression vector of the miR-133b is built by using the pEGFP-C1, and amplification and differentiation of a bovine muscle satellite cell are regulated by the miniature RNA, so as to obtain a high-purity bovine muscle satellite cell. A good cell model is provided for researching the function and the effect of each regulatory factor in amplification and differentiation processes of the muscle satellite cell.

The invention discloses a composition for inhibiting oxidative damage of biological cells and application thereof. The composition comprises vitamin C, vitamin E and R-(+)-lipoic acid, wherein the mass ratio of vitamin C to vitamin E to R-(+)-lipoic acid is 1: (1-3): (1-12). The invention also provides application of the composition for inhibiting free radical oxidation damage of biological cells in non-treatment purpose. According to the composition for inhibiting oxidative damage of biological cells provided by the invention, due to the synergistic effect among the vitamin C, vitamin E and R-(+)-lipoic acid, the composition is capable of improving the antioxidant effect and can be used for inhibiting free radical oxidation damage of biological cells in non-treatment purpose. Specifically, the composition has an antioxidant protection effect for porcine skeletal muscle satellite cells and can achieve an effect of reducing oxidative stress of porcine skeletal muscle satellite cells.