49 results about "Skeletal Muscle Satellite Cells" patented technology

The invention discloses directional knockout on sheep MSTN gene by applying the CRISPR / Cas9 technology, and a method for verifying the impact effect of directional knockout on skeletal muscle satellite cell differentiation. The concrete contents are as follows: firstly, target gene cloning; secondly, gRBA design and synthesis; thirdly, CRISPR / Cas9 gene knockout vector construction; fourthly, CRISPR / Cas9 gene knockout vector exogenous activity detection; fifthly, CRISPR / Cas9 gene knockout vector endogenous activity detection; sixthly, CRISPR / Cas9 gene knockout vector knockout effect detection. The method has the following advantages: firstly, the experimental period is shorter; secondly, the method is simple and practicable, has high repeatability, and can be performed in common laboratories; thirdly, active vector constructed through the method is small in toxic and side effects, and can be applied to the preparation and production of transgenic animals; fourthly, the method is small in non-specific shear, and obviously improves the gene targeting knockout efficiency; fifthly, the method is high in applicability.

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.

The present invention relates to the technical field of molecular genetics, and specifically discloses an application of the pig MDFI gene in regulation of pig skeletal muscle growth and development. According to the present invention, MDFI is overexpressed and interfered in pig skeletal muscle satellite cells, results of cell count and EdU fluorescence detection show that the MDFI can significantly promote proliferation of the pig skeletal muscle satellite cells, and the results of detection on the expression levels of MyoD, myogenin and MyHC expression show that the MDFI can inhibit differentiation of the pig skeletal muscle satellite cells; and with qRT-PCR and Western Blot methods, the regulation effect on the MRFs and the associated cyclin is detected after MDFI is overexpressed and interfered, and the regulation mechanism of the MDFI on the proliferation and differentiation effect of the pig skeletal muscle satellite cells is primarily explored so as to provide the theoretical basis for further research of the MDFI on the pig skeletal muscle growth and development at the body level and provide the reference for improvement of the pork production breeding work.

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 belongs to the technical field of animal genetic engineering, and particularly relates to application of long-chain non-coding RNA IncMGPF in regulation and control of muscle developmentfunctions of pigs. An over-expression vector and interfere vector of long-chain non-coding RNA IncMGPF are constructed, skeletal muscle satellite cells of the pigs are infected at a cellular level ina slow virus infection manner, biceps femoris muscles of the pigs are infected at a living level and are collected after being infected by viruses for multiple times, and the influences of IncMGPF onthe growth and development of the muscles are proved according to the individual pig, so that a technical basis is laid for the cultivation of new species of pork-type pigs with higher yield.

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 belongs to the technical field of genetic engineering and relates to a cell clone with MyoG (myogenin) gene knock-in and MSTN (myostatin) gene knock-out prepared by Crispr / Cas9 technique. The cell clone is characterized in that after being isolated and purified, the cell clone may be induced by 2% horse serum to differentiate into muscle fibers; skeleton satellite cells herein can merge into myotubes; an MSTN gene knock-out vector target is connected successfully, with no base mutations occurring; experimental group cells can differentiate into a certain quantity of myotubes; it is indicated that after MyoG gene is interfered, the cells still can differentiate into myotubes, while total myotube fusion rate is reduced and the form and quantity are significantly differed from those of a control group.

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 belongs to the technical field of screening and application of molecular markers of pigs and specifically relates to a method for increasing muscle mass of the pigs. The method disclosedby the invention increases the muscle mass and muscle fiber density of the pigs through interference and over-expression of an MN864465 gene. The MN864465 gene is obtained through amplification in agenome of large white pigs, wherein a nucleotide sequence of the gene is shown in SEQ ID NO:1, and the sequence is located in areas 104, 826, 111-104, 827 and 563 in a No.8 chromosome of the pigs. Twocarriers are obtained through construction, the gene is over-expressed and intervened in a porcine skeletal muscle satellite cell respectively, and the gene is found to promote proliferation of the porcine skeletal muscle satellite cell and restrain differentiation of the porcine skeletal muscle satellite cell, namely the muscle mass and muscle fiber density of the pigs can be increased. Growth and development of muscle can be restrained during lentivirus injection of leg muscle of the pigs and mice with over-expression of the gene. The method provided by the invention provides new resource for muscle improvement of the pigs.

The invention discloses a feed additive for promoting growth of porcine skeletal muscle and application thereof in the technical fields of feed processing and fish breeding and poultry raising. The feed additive is a carnosine obtained from condensation of beta-alanine and L-histidine with a relative molecular weight of 226.24, and is a natural antioxidant. The feed additive is capable of activating an mTOR signal pathway and enhancing proliferation of porcine skeletal muscle satellite cells, thus promoting the growth of the porcine skeletal muscle. The mechanism of porcine muscle growth is explained in terms of cells and an important idea is offered for development of animal growth promoting additives. When the additive is added to a pig feed, the growth performance of pigs and the meat producing efficiency can be improved. The additive has remarkable economic benefits, is safe without any risk even in long-term use, and has the characteristics of going green, safety, and environmental protection.

The invention belongs to the technical field of molecular genetics, and particularly relates to an lncRNA marker related to porcine skeletal muscle satellite cell proliferation and application of the lncRNA marker. The lncRNA marker is lnc-88672, and a nucleotide sequence of the lncRNA marker is SEQ ID NO.1. It is found for the first time that the expression of the lnc-88672 is related to the porcine skeletal muscle satellite cell proliferation and is continuously reduced along with the continuous increase of the day age of a pig, so that the lncRNA marker related to the porcine skeletal muscle satellite cell proliferation is provided; the porcine skeletal muscle satellite cell proliferation condition can be identified by detecting the expression of the lnc-88672; the inhibition of the expression of the lnc-88672 can promote the porcine skeletal muscle satellite cell proliferation; and a new idea and method are provided for clinical research of porcine skeletal muscle growth and development.

The invention provides a mouse skeletal muscle satellite cell separation and culture method. The mouse skeletal muscle satellite cell separation and culture method comprises following steps: 1, mice are killed through intraperitoneal injection of pentobarbital sodium, and are immersed in 75% alcohol for disinfection; 2, the mice are fixed on plates with the venter posterior surfaces upward, hind limb skin is peeled under aseptic conditions, and thighbone muscle tissues are collected; 3, in an aseptic operation bench, PBS washing is carried out for three times, surface blood vessels, tendons, and connective tissues are removed; 4, the processed tissues are cut into pieces, and are subjected to digestion with preheated trypsin and IV type collagenase respectively; 5, digestion is stopped, centrifugation is carried out, and an obtained supernatant is removed; 6, a cell filtrate is collected, and trypan-blue drying detection is adopted to detect cell activity; 7, after cell counting, a complete medium is adopted for resuspension inoculation into a coated culture flask for culture at 37 DEG C at 5% CO2 environment; 8, differential attachment method is adopted to remove fibrocytes; 9, cell morphology is carried out; 10, ELISA method detection is carried out; and 11, RT-PCR detection is carried out. The mouse skeletal muscle satellite cell separation and culture method is simple in operation, and is high in survival rate; the number of obtained cells is large; the mouse skeletal muscle satellite cell separation and culture method is an ideal method, and is capable of providing experiments with reliable cell resources.

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 embodiment of the invention discloses an isolated culture method of horse skeletal muscle satellite cells. The isolated culture method comprises the following steps: sterilizing horse muscle blocks, removing adipose tissues and connective tissues, and crushing into blocky muscle tissues; digesting the blocky muscle tissue by trypsin, and performing centrifugal separation to obtain a first supernatant and a first precipitate; and centrifuging the first supernatant to obtain a second supernatant and a second precipitate, discarding the second supernatant, re-suspending and centrifuging the second precipitate with a proliferation culture solution to obtain a third supernatant and a third precipitate, discarding the third supernatant, suspending the proliferation culture solution of the third precipitate, filtering through a cell filter, collecting a first filtrate, and centrifuging the first filtrate to obtain a fourth precipitate. According to the embodiment of the invention, the quantity and the purity of the skeletal muscle satellite cells are improved through purification, and an accurate skeletal muscle satellite cell isolated culture method is provided for scientific research colleges and universities for research of muscle growth, muscle injury and the like.

The invention belongs to the technical field of chick embryo hatchery environment illumination, and particularly relates to a method capable of improving the proliferation activity of skeletal muscle satellite cells in the chick embryo hatching period. The method specifically includes the step that illumination with light in different colors is adopted for hatching in the chick embryo hatching period. The light in different colors includes white light, red light, green light and blue light, preferably, green light. The hatching temperature is kept to be 37+ / -0.5 DEG C, and the humidity is kept to be 50%-60%. As for the light regime, 24 h full illumination is carried out, and the illumination intensity is 15 lx. By improving the proliferation activity of skeletal muscle satellite cells in the chick embryo hatching period, the weight of broiler chickens in hatching and the antioxidant level of skeletal muscles after hatching are effectively increased, and the meat quality is 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 discloses an efficient separation and purification method of sheep skeletal muscle satellite cells, which comprises the following steps: soaking muscle tissues in 75% alcohol for 15 minutes, cutting required skeletal muscles, cleaning with PBS (Phosphate Buffer Solution) containing 1% double antibodies, removing connective tissues, cutting the skeletal muscles into 2 mm<3>, transferring the skeletal muscles into a centrifugal tube, repeatedly blowing and beating with the PBS for three times, carrying out natural sedimentation, discarding supernate, transferring tissue blocks into a culture bottle, absorbing the redundant liquid, adding 1ml of culture medium per 25cm<2> growth surface, slowly inclining the culture bottle until tissue blocks are uniformly distributed on the growth surface, covering a bottle cap, culturing at 37 DEG C, 5% CO2 and saturated humidity for 8-24 hours, then adding 1ml of culture medium, continuously adding 1ml of culture medium every 24 hours until the volume of the culture medium is 5ml / 25cm<2>, and changing the culture medium once a week until the cell growth tends to be stable. The method disclosed by the invention is low in cost and extremely small in cell damage, so that more cells can be collected.

The invention discloses a long non-coding RNA marker Lnc-482286 related to porcine skeletal muscle satellite cell proliferation, and application of the long non-coding RNA marker Lnc-482286, and belongs to the technical field of molecular biology. The long non-coding RNA marker is Lnc-482286, and the nucleotide sequence of the long non-coding RNA marker Lnc-482286 is shown as SEQ ID NO.1. The marker Lnc-482286 provided by the invention is highly expressed in porcine muscle tissues, expression of the Lnc-482286 in the muscle tissue of the Jinfen white pig is proved to be in a trend of firstly increasing and then decreasing along with the increase of the day age, and the Lnc-482286 plays a significant up-regulation role in the process of the porcine skeletal muscle satellite cell proliferation. The proliferation condition of the porcine skeletal muscle satellite cells can be identified by detecting the expression of the Lnc-482286, the porcine skeletal muscle satellite cell proliferation can be accelerated by improving the expression of the Lnc-482286, and a new thought and method is provided for clinical research on the growth and development of the porcine skeletal muscle.

The invention relates to an application of beta-N-acetylglucosaminyl transferase to cold-resistant medicine preparing. According to the application, the OGT expression quantity and the time of cold stress are positively related, the OGT expression quantity after the cold stress is increased, it can be protected that skeleton satellite cells are not damaged by the cold stress, and the cold-resistant effect on skeletal muscle can be achieved; OGT is injected into the bodies of mice, and the cold-resistant capacity of the mice can be obviously improved; in the way, the OGT can serve as cold-resistant medicine to be popularized in a large-scale mode.

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.