73 results about "Fibroblast cell line" patented technology

The invention discloses a culture solution for an in vitro efficient amplification of animal cells and an application of the culture solution. The culture solution consists of basal culture mediums, namely DMEM / F12, bFGF, EGF, sodium pyruvate, glutamine, sodium hydrogen carbonate and fetal calf serum. The culture solution disclosed by the invention has comparatively low content of animal serum, and is comparatively high in safety, simple and definite in formula components; the culture solution can avoid a possibility of different properties of the cultured cells due to a difference between culture medium batches, and is applicable to industrial production of cell products. Animal adherent cells cultured by the culture solution disclosed by the invention are comparatively strong in in-vitro amplification capacity and well maintain biological characteristics; and the culture solution is not only applicable to culture of adherent mesenchymal stem cells, but also suitable for culture of other fibroblast lines and other cell lines.

The present inventors discovered that although suppressing expression of the RecQ1 gene, a RecQ helicase family gene, shows suppressive effects on cell proliferation in cancer cells, such effects are not observed in human TIG3 cells (a normal diploid fibroblast cell line), which are normal cells. Hence, the present inventors discovered that siRNAs against RecQ1 gene have cancer cell-specific cell proliferation-suppressing effects that are mediated by suppression of the expression of said gene.

The invention belongs to the technical field of animal genetic engineering and particularly relates to a method for improving pig meat quality. The method is characterized in that a peroxisome proliferator-activated receptor (PPAR) gamma gene serves as an important candidate gene for improving the meat quality, a fibroblast cell line of a 30-day-old fetus of a large white pig is established, a SwaI linearized pN1-MCK-PPAR gamma2 expression vector is shifted to the 30-day-old fetus fibroblast cell line of the large white pig through an electrotransfection method, and a PPAR gamma transgenic pig is prepared in a method of somatic nucleus transplantation. The influence of muscle tissue overexpression PPAR gamma genes on meat traits such as intramuscular fat deposition is verified in a transgenic pig, the contradiction of simultaneously selecting meat quality and meat quantity in conventional animal breeding is overcome, and the novel method is provided for cultivating lean meat pigs with good meat quality.

The invention relates to a method for constructing a Tibetan antelope skin fibroblast line, which comprises the following steps of: (1) sampling; (2) carrying out primary culture of Tibetan antelope ear skin tissue; (3) carrying out passage and purification; (4) carrying out cell cryopreservation; and (5) carrying out cell recovery. By establishing the method for primary culture, cell passage, cell cryopreservation and cell recovery, the invention obtains a high-activity Tibetan antelope fibroblast line, and provides research materials and technical support for further developing related researches of Tibetan antelope molecular biology and cell biology.

The invention belongs to the technical field of biology, and particularly relates to a micropterus salmoides myocardial fibroblast cell line and application. According to the cell line, aiming at thecharacteristics of micropterus salmoides heart tissue cells, brain tissue cells are separated by adopting a collagenase I and pancreatin combined digestion method, primary culture is performed, the micropterus salmoides myocardial fibroblast cell line is successfully constructed and is continuously passed to more than 60 generations, a large number of micropterus salmoides heart-derived cells canbe provided, and the cells can maintain a good growth state and can be cryopreserved. The micropterus salmoides myocardial fibroblast cell line has sensitivity to various fish viruses, cytopathy is generated after virus inoculation, the cell line can be directly applied to pathogen characteristic research and vaccine development, and the micropterus salmoides myocardial fibroblast cell line can also be used for expressing exogenous genes for gene function research.

The invention belongs to the technical field of biology, and discloses double sgRNA which is a combination of one of STNG-sgRNA 1-3 targeting a fourth exon and one of STNG-sgRNA 4-5 targeting an eighth exon. After the double sgRNA is carried on an expression vector, an appropriate gene knockout vector can be obtained, the STING gene of the porcine fibroblast is knocked out in a large fragment mode, and therefore the obtained porcine fibroblast line has the advantages of being suitable for cloning, free of knockout gene repairing capacity and free of exogenous gene introduction. Meanwhile, the invention also discloses a construction method of the porcine fibroblast line.

The invention discloses a cow fetus fibroblast cell containing an Iprl macrophage metamerism expression vector. A cow fetus fibroblast cell line which contains an Iprl macrophage metamerism expression vector Psp-EGFP-Iprl, is based on the vector and contains the Iprl macrophage metamerism expression vector is established, and the cell line is used as a nucleus donor cell, which provides massy foundation for transgene cow with tuberculosis. In the invention, an exogenous expression vector Psp-EGFP-Iprl is transduced to the cow fetus fibroblast cell through an electrotransfection method, the expression condition of a marked gene EGFP is observed by a fluorescence microscope, positive cells are selected by G418, the target gene Iprl is confirmed to be integrated into the genome of the cow fetus fibroblast cell by PCR identification, and finally the cow fetus fibroblast cell subjected to the transfection of the Iprl gene is moved into cow denucleated oocyte as a donor to obtain a transgene clone embryo.

The invention discloses a trachemys scripta elegans embryo fibroblast cell line and a construction method thereof. The construction method comprises the following steps: taking a trachemys scripta elegans egg hatched for 12 to 18 days, selecting a live embryo and then sterilizing the trachemys scripta egg; shelling, taking the trachemys scripta embryo, discarding the head, fours limbs and visceraof the trachemys scripta embryo, and flushing the rest embryonic tissues with a PBS (Phosphate Buffered Saline) containing double-antibody until no obvious bloodstain is found; using trypsin to digestthe embryonic tissues which are flushed until no obvious bloodstain is found, collecting digested monoplast, and inoculating the monoplast into a complete medium for static culture; when the cells are partially adherent and do not float by slightly oscillating, absorbing the complete medium and the cells which are not adhered, and adding the rest adherent cells into a new complete medium for continuously culturing; when the confluence of primary cultured cells reaches 90 percent or above, digesting with the trypsin, collecting the monoplast and then inoculating into the complete medium for subculture. The invention lays a material and technical foundation for further using RSTEFs for conducting basic theory and experimental research.

The invention provides a method for culturing human induced pluripotent stem cells by using human urine cells as a feed layer. Third-twelfth generations of human urine cells cultured after subculture,cryopreservation and resuscitation are used as trophoblast cells for culturing the human induced pluripotent stem (hiPS) cells reassembled by adult male foreskin fibroblasts. The used human urine cells replace small mouse embryo fibroblasts or mouse embryo fibroblast cell lines used in traditional methods, the human induced pluripotent stem cells are cultured, pollution of heterologous cells in the culture system is reduced, and the human induced pluripotent stem cells are more easily obtained than mesenchymal stem cells. The culture system is proved to make the human induced pluripotent stemcells amplified in vitro, the biological characteristics and the pluripotent property of the hiPS cells are maintained for a long time, and the possibility is provided for the hiPS to enter the clinical application.

The invention discloses a breeding method for transgenic pigs expressing sIFITM3 (swine interferon induced transmembrane 3) genes. The breeding method comprises the steps as follows: firstly, the construction of swine fibroblast cell lines stably expressing the sIFITM3 genes comprises the following steps: the step a refers to the construction and the linearization of pcDNACAsIITM3 vectors, wherein, primers are designed, lymph node tissue RNA (Ribose Nucleic Acid) of pigs is extracted, the sIFITM3 genes are obtained through RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification, ethanol precipitation is carried out, and sterile water is used for dissolving; the step b refers to liposome transfection, wherein, swine fibroblasts expressing sIFITM3 are constructed; the step c refers to screening and proliferation of nuclear donor cells transfected with the sIFITM3 genes; secondly, recombination blastula acquisition based on a manual nucleus transplantation method comprises the following steps: the step a refers to the preparation of recipient cells; the step b refers to enucleation and injection of the nuclear donor cells; and thirdly, the embryo transplantation and the acquisition of the transgenic pigs comprise the following steps: the step a refers to embryo transplantation, wherein, bred blastulas are transferred to a fallopian tube of a surrogate sow, and then detection is carried out; and the step b refers to transgenic individual identification. The breeding method is feasible, and is simple and convenient to operate; in addition, the pigs are the transgenic pigs expressing sIFITM3, and have the potential capability to resist animal virus such as foot-and-mouth disease virus, Japanese encephalitis virus, swine influenza virus and the like.

The invention belongs to the field of cell biology. A morphologically uniform and vigorously-growing fibroblast cell line of Hubei white pig fetus is prepared through the following steps: taking a Hubei white pig fetus which has been in pregnancy for 35 days as the raw material, then subjecting the fetus to treatments of mechanical shearing and pancreatin digestion, and finally subjecting the digestion product to processes of primary culture, subculture, liquid nitrogen freezing storage, and cell thawing so as to obtain the finished product; the fibroblast cell line is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC No. C201331. The obtained fibroblast cell line of Hubei white pig fetus has a high purity, a uniform shape, and no compounding of epithelial cells, liver cells, myocardial cells, and the like. The cell line provided by the invention can provide high quality cell materials for gene engineering, cell engineering, embryo engineering, immunology and molecular biology, can be used as donor cells for porcine somatic cell nuclear transplantation, is an important genome resource bank, and is a species preservation method for local excellent variety.

The invention discloses a macrophage specific targeting vector and a recombinant cell constructed based on an Ipr1 gene. According to the invention, an MSR1 promoter is connected to a 5' terminal of the Ipr1 gene, such that the Ipr1 gene is specifically expressed in bovine macrophage with high efficiency; a recombinant gene LA-MSR1-Ipr1-SA is further constructed, such that a target gene is subject to site-directed integration, and is integrated between bovine 28-chromosome SP-A and MAT1A gene. An Ipr1 macrophage specific targeting vector pLMIS is constructed, and is transfected into newborn bovine ear fibroblast cells; through G418 and GCV positive and negative screening, transgenic newborn bovine ear fibroblast cell lines are constructed; the transgenic newborn bovine ear fibroblast celllines are adopted as nuclear donors, and are transplanted into bovine enucleated oocytes, such that transgenic cloned embryos are obtained; when the embryos are transplanted, five cattle with site-directed inserted Ipr1 gene is produced, and four of them survive. The transgenic cattle is resistant to M.bovis.

The invention discloses a high-living rate and high-purity mature fiber cell system of Mongolian horse (CGMCC No.1879) in the cellar biological domain, which is characterized by the following: culturing high-purity phoroblast without epithelial cell; maintaining the living rate of frozen cell between 93.2% and 98.7%; fitting for large scale of culturing.

The invention relates to the technical field of genetic engineering, and especially relates to a sgRNA for targeted knockout of a FRZB gene, a porcine embryo fibroblast cell line for knocking out theFRZB gene and applications of the fibroblast cell line. The provided double sgRNA can cut two loci on the first exon of the FRZB gene, then after the sgRNA is constructed into an expression vector, then porcine embryo fibroblast cells are transfected through an optimized electro-transformation system, and a flow cytometer is used to sort positive monoclonal cells with high purity, so that the FRZBknocked-out cell line can be obtained through genome identification and screening. Thus, the problems of low transfection efficiency, low targeting efficiency and low monoclonal purity during the construction of knockout cell lines can be solved; and simple operation can be realized, and the edited cell line with longer fragment deletion can be obtained. The related cell line can be used as the donor of nuclear transfer of somatic cells for the preparation of transgenic swine, so that good research tools can be provided for further exploring the biological functions of the FRZB gene.

The invention provides a method for culturing goose parvovirus by using a goose embryo fibroblast line, belongs to the field of veterinary biological products, and solves the problem of low production efficiency of an existing goose parvovirus culturing method. The method comprises the following steps: culturing primary cells, namely cutting a goose embryo which well develops into small pieces, washing, digesting, blowing and dispersing digestive cells to obtain a cell suspension, culturing until the cells form complete monolayers so as to obtain the primary cells; establishing a goose embryo fibroblast line, namely removing a nutrient solution from the primary cells of the goose embryo fibroblast, washing, digesting and culturing until complete monolayer cells are formed to obtain F1-generation cells which can be passed by three generations; reproducing and harvesting, namely inoculating F1-F4-generation cells which form 70 percent of monolayer, and harvesting reproduced viruses when over 75 percent of cells is diseased. According to the method, the amount of harvested virus liquid is 255 times of the dose of primary cell inoculation culturing, and the virus titer of the virus liquid is 10-100 times higher than that of a virus liquid which is inoculated, cultured and harvested when 100 percent of monolayer is formed.

The invention relates to an orthopedic implant with a nano blind hole structure on the surface, wherein the orthopedic implant surface is provided with the nano blind hole structure, the uppermost surface of the orthopedic implant is a nano blind hole layer, the middle thereof is a TiO2 blocking layer, and the lowermost surface thereof is a Ti basal layer. The invention also provides application of the orthopedic implant with the nano blind hole structure on the surface. The invention has the advantages that: the nano blind hole structure is manufactured on the orthopedic implant surface by utilizing a nanotechnology, and the orthopedic implant with the nano blind hole structure has a promoting action on the adhesion proliferation and the differentiation of a mouse C3H10T1 / 2 fibroblast cell line with a multi-directional differentiation potential; and meanwhile, the surface also has a remarkable inhabiting action on staphylococcus epidermidis ATCC 35984 with a positive biomembrane and shows a certain antibacterial action. In sum, the orthopedic implant with the nano blind hole structure on the surface can improve the osseous integration and the antibacterial performance of an orthopedic metal implant material surface.

The invention relates to an ungulate animal cell line for an inductively expressed pluripotent maintenance gene and construction thereof. The ungulate animal fibroblast cell line for expressing and maintaining pluripotent key gene oct4 by an inducible system (tet-on) is established by the invention. The cell line has the characteristics of low virus copy number, obviously up-regulated oct4 expression and high purity for cell single cell source. The cell line is used as donor cell for nuclear transplantation; and the expression of oct4 is induced by drug doxycycline, thereby facilitating reprogramme of egg cells after the nuclear transplantation is facilitated and finally achieving the object of establishing embryonic stem cells of the ungulate animals, so as to breed various transgenic animals.

The present invention is high activity and high purity fibroblast line of Minxian county black fur sheep obtained with the ear margin tissue and through primary cell culture, subculture and cell freezing preservation and in the preservation number of CGMCC No.1880, and belongs to the field of cell biology. The high activity and high purity fibroblast line is suitable for great scale culture. The present invention makes it possible to provide great amount of high quality fibroblast line of Minxian county black fur sheep for the scientific research in gene engineering, cell engineering, etc.

The invention provides a preparation method of an MC3R gene edited porcine fibroblast line, and belongs to the field of gene edition. The preparation method comprises the following steps: constructinga gene knockout vector of which the nucleotide sequence of sgRNA is shown as SEQ ID NO: 1 and a gene knockout vector of which the nucleotide sequence of sgRNA is shown as SEQ ID NO: 2, establishing aporcine fibroblast line, transfecting the porcine fibroblast line with the gene knockout vector, and performing screening and positive cloning identification to obtain MC3R gene homozygous knockout monoclonal cells. The preparation method of the MC3R gene edited porcine fibroblast line provided by the invention has the advantages of simplicity, quickness, high efficiency and lower cost.

The invention relates to a method for establishing and culturing Tianzhu Yak'ear marginal fibroblast cell line, utilizing the fast tissue wall pasting method to improve culture medium, and finally obtaining high-yield and high-purity Tianzhu Yak'ear marginal fibroblast cell line whose conservation number is CGMCC No.1881, belonging to the field of cell biology. And the frozen cells have stable quality, suitable to large scale culture. And the Tianzhu Yak'ear marginal fibroblast cell line can provide large numbers of high quality materials for research on science of living systems in gene engineering, cell engineering, immunity and molecular biology; can act as donor cells for animal's somatic cell cloning and breeding; and can enrich local variety improvement and storage of local excellent variety in agriculture.

The invention relates to the technical field of cell genetic engineering, in particular to preparation and application of a porcine embryo fibroblast cell line edited by a CD163 gene. The pig embryonic fibroblast cell line can reduce carrying of exogenous hereditary materials and can reduce other biological function loss; the preparation comprises the following steps of (1) expression of Cas9 protein, (2) purification of Cas9 protein, (3) design of an sgRNA leader sequence, (4) in vitro transcription, (5) porcine embryo fibroblast nuclear transformation, (6) cell dilution, (7) separated screening, (8) expanded culture and (9) collection and identification. The cell line can be used as a donor of somatic cell nuclear transplantation for the preparation of transgenic pigs.

The invention discloses a method for constructing a 14-3-3 epsilon gene knockout cell strain based on a CRSIPR technology and application of the 14-3-3 epsilon gene knockout cell strain, and belongs to the technical field of biology. The invention provides an sgRNA sequence with 14-3-3 epsilon gene knocked out. The nucleotide sequence of the sgRNA sequence is as shown in SEQ ID NO: 1. The 14-3-3epsilon gene is knocked out on a chicken fibroblast line DF-1 cell genome for the first time by utilizing a CRISPR-Cas9 technology, so that the expression of 14-3-3epsilon protein is completely lost, and the 14-3-3epsilon knocked-out DF-1 cell strain is obtained; wherein the activity, the growth speed and the like of the knockout cell strain are not obviously different from those of a control cell, and the DF-1 knockout cell model is an ideal DF-1 knockout cell model. The method is simple and convenient to operate, short in period, low in cost and stable in cell strain after modification, and can be used for researching the biological function of the 14-3-3 epsilon protein.

The invention relates to the technical field of genetic engineering, and especially relates to a sgRNA for targeted knockout of a TNF[alpha] gene, a porcine embryo fibroblast cell line for knocking out the TNF[alpha] gene and applications of the fibroblast cell line. The target sequence of the provided sgRNA on the TNF[alpha] gene is located on the first exon and shown as SEQ ID NO: 1. After the sgRNA is constructed into the vector px330, an expression vector pX330-TNF[alpha] is taken as the targeting vector of the TNF[alpha] gene; and porcine embryo fibroblast cells are transfected so as to obtain the TNF[alpha] gene-knocked-out monoclonal cell line of porcine embryo fibroblast cells, and the obtained mutant having many different editing types can cause frameshift mutation, so that high editing efficiency can be achieved. The prepared cell line can be used as the donor of somatic cell nuclear transfer for the preparation of transgenic swine, so that the cell line has large applicationand research values.

Palladium (II) N-heterocyclic carbene (NHC) complexes which are stable in the presence of biological thiols. A representative complex, [Pd (C^N^N) (N, N' -nBu 2NHC) ] (CF 3SO 3) (Pd1d, HC^N^N= 6-phenyl-2, 2 ' -bipyridine, N, N'-nBu 2NHC=N, N'-di-n-butylimidazolium), displays potent killing activity toward cancer cell lines but is less cytotoxic toward normal human fibroblast cell lines.

Aspects of the invention provide an immortalized fibroblast cell line capable of the growth and maintenance of human embryonic stem cells. An immortalized fibroblast cell line derived from an early stage mouse embryo is provided. Methods of preparing an immortalized fibroblast cell line from an early stage mouse embryo are also provided.

The invention relates to a method for producing a parthenogenetic cloned pig by means of continuous cloning technology, which belongs to the field of reproductive biology. The method provided by the invention comprises the following steps of: the degreasing of a porcine oocyte, parthenogenetic activation, the separation of a fibroblast, and continuous somatic nuclear transfer; and specifically, the method comprises the following steps of: carrying out the parthenogenetic activation on the degreased mature porcine oocyte to obtain a parthenogenetic embryo, transplanting the parthenogenetic embryo into the body of a surrogate sow to obtain a 25-29 days old living parthenogenetic fetus, establishing a porcine parthenogenetic fetus fibroblast line which is used as a preliminary donor cell, and carrying out continuous cloning by means of somatic cell nuclear transfer technique to obtain a porcine parthenogenetic individual. By adopting the method provided by the invention, the condition that the in-vivo development stops when the porcine parthenogenetic fetus grows for 30 days is overcome, the in-vivo development time of the porcine parthenogenetic fetus is successfully prolonged, and therefore a living parthenogenetic cloned pig can be successfully obtained finally.

The invention provides a method for culturing duck plague virus, which belongs to the technical field of virus culture. The invention adopts chicken embryo fibroblast cell line DF-Duck plague virus (DPV) was adapted to DF by inoculating DPV with overdose and adding chicken embryo allantoic fluid (CEAF) in the cell fluid, and was passed blindly from generation to generation. 1 cell line and producecytopathic effect. The duck plague virus culture method of the invention is simple in operation, and DF-1 Duck plague virus (DPV) grew well in cell line culture, the virus content was 107.4-108.0 TCID 50 / ml, which was 0.25% higher than that in CEF cell culture. 0.75 titer, avoiding the potential safety hazard of exogenous virus contamination in CEF cell culture, and due to DF-After the establishment of the cell library, the stability and sustainability of the production are ensured, and the production cost of duck plague virus in the process of culture is greatly reduced, which can improve the safety and reduce the cost, and the economic benefit is remarkable.