31 results about "Lipofection" patented technology

The construction of transgenic animal models of human Alzheimer's disease, and methods of using the models to screen potential Alzhe## disease therapeutics, are described. The models are characterized by pathologies similar to pathologies observed in Alzheimer's disease, based on expression of all three forms of the β-amyloid precursor protein (APP), APP695, APP751, and APP770, as well as various point mutations based on naturally occurring mutations, such as the London and Indiana familial Alzheimer's disease (FAD) mutations at amino acid 717, predicted mutations in the APP gene, and truncated forms of APP that contain the Aβ region. Animal cells can be isolated from the transgenic animals or prepared using the same constructs with standard techniques such as lipofection or electroporation. The transgenic animals, or animal cells, are used to screen for compounds altering the pathological course of Alzheimer's disease as measured by their effect on the amount of APP, β-amyloid peptide, and numerous other Alzheimer's disease markers in the animals, the neuropathology of the animals, as well as by behavioral alterations in the animals.

The invention relates to compositions and methods for a one-step synthetic technique for making cationic steroid or cationic drug molecules for use as delivery vehicles. The invention further relates to methods for using cationic steroid molecules in lipofection or transfection, delivery of drugs, and for treatment of inflammation and other diseases and disorders. The invention also relates to cationic steroid prodrugs and cationic prodrugs and to methods of modifying drugs.

The invention discloses a method for enhancing mesenchymal stem cell chemotactic capacity and chemotactic factor CCL5 expression, and relates to mesenchymal stem cells. The method comprises the steps of separation of the mesenchymal stem cells and passage of primary culture umbilical cord mesenchymal stem cells; identification of the umbilical cord mesenchymal stem cells; stimulation of TNF-alpha and IFN-gamma to hUC-MSCs; detection of CCL5 expression through Real-Time PCR; detection of CCL5 expression through ELISA. CCL5 expression through MSCs is improved by 924 times, a large amount of natural CCL5 can be obtained, no lipofection transfection is needed, and rapidness and convenience are achieved. Due to simulation of TNF-alpha and IFN-gamma, the transfer ability of the mesenchymal stem cells is improved, it is promoted that the mesenchymal stem cells can more rapidly collect tissue injuries and inflammation portions in vivo, and the functions of tissue repairing and immune modulating are brought into play.

The invention belongs to the technical field of pig transgenosis and particularly relates to a method for producing transgenic pigs through overexpression HOXA10 genes. The method is characterized in that constructed HOXA10 gene expression carriers are subjected to lipofection and sulfate (G418) screening to obtain HOXA10 transgenic cell lines with stable expression; the HOXA10 transgenic cell lines are transplanted into denucleated pig oocytes with HOXA10 transgenic pig fibroblasts as nuclear donors through an artificial nuclear transplantation method to form reconstructed embryos; the reconstructed embryos are transplanted into a receptor sow oviduct, and after a sow gives birth to the HOXA10 transgenic pigs and molecular identification is conducted, masculine HOXA10 transgenic pigs are screened out. The transgenic pigs obtained through the method provide materials for research about the mechanism of improving the reproductive capacity through the HOXA10 genes and provide an animal model for revealing other functions of the HOXA10 genes.

The invention relates to a cellular expression system for stably expressing an anti-TNF-alpha monoclonal antibody, and a construction method and application of the cellular expression system. According to the cellular expression system, light and heavy chain encoding genes of the anti-TNF-alpha monoclonal antibody are optimized according to cell codon preference so as to construct anti-TNF-alpha human-mouse chimeric monoclonal antibody homologous recombination vector with a Bak1 gene and the like as a drone, meanwhile, a CRISPR / Case9 vector with a specific cutting effect is constructed for exon regions of drone genes such as Bak1, two vectors are commonly guided into Chinese hamster ovary cells (CHO) by lipofection transfection, and a positive cell strain capable of stably expressing the anti-TNF-alpha monoclonal antibody is obtained through screening. The cellular expression system can be applied to efficient production of the anti-TNF-alpha monoclonal antibody, and the anti-TNF-alpha monoclonal antibody can be used for treating diseases such as rheumatoid arthritis and ankylosing spondylitis.

Provided is an efficiency improving agent for gene transfer to mammalian cells, a method for improving efficiency of gene transfer to mammalian cells, and a method for transforming mammalian cells. The method is characterized in that tRNA is used in combination with a lipofection reagent. Preferably, the agent may be used so that the tRNA concentration in a lipofection solution falls within the range of 3 to 50 μg / mL, and the concentration in a culture is approximately 1 / 10. More preferably, tRNA and PEG may be used in combination with a lipofection reagent. According to the present invention, gene transfer to mammalian cells with high efficiency can be achieved.

The invention provides a method for preparing an immortalized duck embryo liver cell line, the method comprising: (1) digesting the duck embryo liver with a trypsin digestion method, cultivating the digestive juice, and obtaining primary cultured duck embryo liver cells; (2) Using liposome transfection method, the eukaryotic expression plasmids encoding hTERT and Marker genes were introduced into the primary cultured duck embryo liver cells; (3) The transfected duck embryo liver cells were cultured, and the G418 was used to screen the cells hTERT-positive clones; (4) culturing the screened hTERT-positive clones for more than 50 passages to obtain immortalized cell lines. The cell line still has good vigor after more than 50 passages in vitro, maintains division and proliferation, does not undergo senescence and apoptosis, and is an immortalized cell line. The immortalized duck embryo hepatocyte has regular shape, simple culture conditions, easy control and convenient use, and provides a good carrier for studying duck diseases, especially duck hepatitis virus.

The invention relates to a method for constructing hTERT mediated T lymphocytic model and a cell bank thereof in the field of medicines. The method is mainly characterized by comprising the following steps: carrying out double enzyme digestion on a plasmid pCIneo-hTERT and a carrier pLXSNneo with incision enzymes EcoR I and Xho I, connecting hTERT and pLXSNneo digested products subjected to PCR amplification and gel electrophoresis separation by virtue of Ligation Mix to construct a pLXSNneo-hTERT recombinant, transforming DH5a competent cells for purifying, amplifying and selecting ampicillin-resistant bacterial colony extraction plasmids, and performing in-vitro subculture on T lymphocytes in logarithmic growth by virtue of lipofection transfection, so that the recombinant is integrated with DNA of cells; performing enlarged culture, performing G418 screening on cells containing positive recombinants, screening cells of which the cellular morphology, growth curve, karyotype, inoculated nude mice test, transfection cell telomerase activity, hTERT mRNA expression, immunohistochemistry, and cell generation cycle and apoptosis rate accord with the characteristics of immortalized cells and which are the same or similar to primary cells to serve as an hTERT mediated in-vitro T lymphocytic research model so as to be cryopreserved in liquid nitrogen. Therefore, a foundation is laid for long-term in-vitro research of pathogenesis of related diseases from the cellular level.

The invention provides a VP2 gene and NP gene recombinant adenovirus and application thereof. The preparation method comprises the following steps: respectively amplifying VP2 genes and NP genes fromSD strains of IBDV (Infectious Bursal Disease Virus) and AIV (Avian Influenza Virus), cloning the obtained target genes into shuttle vectors pDC315-MCS-EGFP, performing co-transfection on a recombinant adenovirus shuttle plasmid pDC315-VP2-NP-EGFP, an adenovirus macroskeleton pBHGlox(delta)E1 and 3Cre in HEK293 cells by utilizing a lipofection transfection method, recombining and packaging, thereby obtaining the recombinant adenovirus pBH-VP2-NP-EGFP containing the VP2 genes and NP genes. The recombinant adenovirus is capable of stimulating the body to produce an antibody, the IBDV antibody content determined by an agar diffusion test reaches an effective antibody level, and the AIV antibody content determined by a hemagglutination inhibition test method is obviously superior to that in acontrol group pBH-EGFP; and the challenge test results show that the protection rate reaches 90%.

The invention relates to construction of an hTERT mediated trisomy 18 syndrome cell model and a cell bank thereof in the medical field. The construction is mainly characterized in that incision enzymes EcoR I and Xho I are used for carrying out double enzyme digestion on plasmid pCIneo-hTERT and carrier pLXSNneo, and Ligation Mix is used for connecting hTERT and pLXSNneo enzyme-digested products subjected to PCR amplification and gel electrophoresis separation to construct a pLXSNneo-hTERT recon; DH5a competent cells are transformed to purify, amplify and pick ampicillin-resistant bacterial colony extraction plasmids; trisomy 18 syndrome cells in logarithmic growth during in-vitro passage are subjected to lipofection transfection; the recons are integrated with cell DNAs and are subjected to amplification culture; positive recon colons are subjected to G418 screening; cells, which meet the immortalized cell characters in the aspects of cellular morphology, growth curve, karyotype, nude mice tumorigenesis test, transfected cell telomerase activity, hTERT mRNA expression, immunohistochemistry, and cell generation cycle and apoptosis rate and are as same as or similar to primary cells, are screened to be as the hTERT mediated trisomy 18 syndrome external research cell models to be frozen in liquid nitrogen. Therefore, the foundation of in vitro long-term research of pathogenesis of trisomy 18 syndrome based on the cellular level is established.

The invention discloses a method for constructing a recombinant baculovirus for expressing L-amino acid oxidase of the macula macula. The gene whose DNA sequence is shown in SEQ ID NO: 1 is cloned into the vector pMD18-T to construct a recombinant plasmid; Restriction endonucleases EcoRI and XhoI cut the recombinant plasmid and the donor plasmid respectively, and ligated the digested products to obtain the recombinant donor plasmid; the recombinant donor plasmid was transformed into E. coli, so that the target gene and the bacillus contained in E. The virus shuttle vector is specifically transposed, and the recombinant bacmid is screened and extracted; the recombinant bacmid is transfected into insect cells by liposome transfection method, and the recombinant baculovirus is harvested after the insect cells are pathological.

The invention discloses a method for detecting CHO cell proliferation by using optical analysis and application thereof. The method comprises the following steps: (1) converting pEGFP-N2 into Escherichia coli DH5alpha, shaking the bacteria, and extracting endotoxin-free plasmids; (2) inoculating CHO cells into a pore plate, performing transient transfection on the plasmids in the step (1) into the CHO cells by adopting a lipofection transfection method when the cell growth density is 70 percent; (3) screening the transfection cells by adopting geneticin G418, performing resistance screening of over 30 generations at least, thereby obtaining a cell strain which can stably express the GFP; and (4) performing multiplication culture on the cell strain which can stably express the GFP, digesting the cells, performing cell counting by adopting trypan blue, detecting the fluorescent intensity by adopting an enzyme linked immunosorbent assay detector, and judging the CHO cell proliferation conditions based on a linear relation between GFP fluorescent intensity in the cells and the cell number. The method disclosed by the invention has the advantages of operability, detection result accuracy, high repeatability, high sensitivity, non-toxicity and the like.

The invention discloses a pair of Talen recognition sequences targeting the zebrafish Forkhead box n1 gene and a method for preparing mRNA for knocking out the zebrafish Forkhead box n1 gene. The gene sequences of the Talen recognition sequence pair are respectively shown in the sequence table SEQ ID NO.1 and SEQ ID NO.2, and the preparation method of the mRNA comprises the following steps a) assembling and synthesizing the Talen recognition sequence described in claim 1 Right; b) according to the Talen recognition sequence pair described in a), construct an expression vector targeting the gene knockout effector protein Talen of the Forkhead box n1 gene; c) transfect the vector obtained in step b) by liposome, Detecting protein expression in 293T cells; d) transcribing the vector obtained in step b) in vitro to obtain the mRNA for targeted knockout of the zebrafish Forkhead box n1 gene. The mRNA obtained by the method of the present invention can be used to knock out the zebrafish Forkhead box n1 gene to prepare a research model of zebrafish thymus development. Compared with the prior art, the method has the advantages of low cost, simple design, high success rate and obvious effect.

The invention relates to a method for establishing a congenital heart disease ventricular septal defect cell model by virtue of a recombinant hTERT and a cell bank of the congenital heart disease ventricular septal defect cell model, belonging to the field of medicines. The method is characterized by comprising the following steps: carrying out double-digestion on plasmid pCIneo-hTERT and a vector pLXSNneo through endonucleases EcoR I and Xho I, connecting Ligation Mix with a digestion product of hTERT and pLXSNneo, which is subjected to PCR amplification and gel electrophoresis separation, thus establishing a pLXSNneo-hTERT recombinant, transforming DH5a competent cells so as to purify, amplify and extract the plasmid, carrying out lipofection transfection on congenital heart disease ventricular septal defect cells of in vitro passage and in logarithmic growth, integrating the recombinant with cell DNA, carrying out amplifying culture, screening cells with positive recombinants by using G418, cloning, screening cells of which the cellular morphology, the growth curve, the karyotype, the inoculated nude mice experiment result, the transfection cell telomerase activity, the hTERT mRNA expression, the immunohistochemistry, the cell generation cycle and the cell apoptosis rate meet the characteristics of immortalized cells and are identical or similar to those of primary cells as an hTERT transfected congenital heart disease ventricular septal defect cell model, and freezing and storing the cell model in liquid nitrogen. A foundation is laid for studying the pathogenesis in vitro for a long time at the level of cells is made.

The invention relates to construction of an SV40LT gene-mediated megakaryocyte model and a cell bank of megakaryocyte, belonging to the field of medical research. The construction is mainly characterized in that an SV40LTag-pcDNA3.1 (-) recombinant is constructed from T4DNA ligase, BamHI, pcDNA3.1 (-) DNA and SV40LTag DNA by virtue of conventional means; the recombinant is purified by virtue of competent escherichia coli; the recombinant is introduced into megakaryocyte cultured in vitro through a lipofection transfection method, so that the recombinant is integrated with the DNA of the cell; a cell screened by virtue of G418 and containing a positive recombinant is subjected to subculture and enlarged cultivation; a cell which is consistent with the biological properties of an immortalized cell in cellular morphology, cell growth curve, karyotype, nude mice carcinogenicity test, detection of SV40 large T gene in transfection cell DNA, determination of an mRNA expression product and a DNA sequence determination result is screened out as a megakaryocyte model, and is cryopreserved in liquid nitrogen, so as to construct an SV40LT gene-mediated megakaryocyte model and the cell bank of megakaryocyte; therefore, a method for long-term in vitro subculture of the megakaryocyte is established, and the method is applicable to the in vitro research of development, function and pathogenic mechanism of the megakaryocyte.

The invention discloses a recombinant plasmid PEGFP-12 obtained by mutating mycobacterium tuberculosis ERP 12 sequence motif to PGLTS. According to a sequence of mycobacterium tuberculosis wild ERP, an ERP complete sequence of which the 12 sequence motif is fully mutated to PGLTS is synthesized, is optimized by way of humanization to be constructed to a PEGFP-N1 eukaryotic expression vector, and the obtained product is named PEGFP-12. The sequence after mutation is shown in SEQ ID: NO: 1. According to the invention, the recombinant plasmid PEGFP-12 of which mycobacterium tuberculosis ERP 12 sequence motif is mutated to PGLTS is constructed. The plasmid is transferred to the A549 cell by virtue of a lipofection transfection method, then protein chip detection is carried out and the sequence is compared with the wild ERP sequence, and after the sequence motif is fully mutated to 12 PGLTS, expression of cell inflammation related factors is obviously up-regulated. Therefore, a relatively obvious reaction can be induced after mutation, thereby providing powerful data for researching PGLTS sequence motif mutation and functional relationship.

The invention discloses novel application of a pig GADD45a gene and construction and application of a high-expression cell line. A method comprises the following steps: according to a full-length geneof a pig GADD45a gene and a vector pLEX-MCS, designing and synthesizing an amplification primer which comprises digestion loci and the full-length gene of GADD45a; performing amplification from piglet fat tissue so as to obtain the full-length gene of the GADD45a gene, performing recycling and digestion, connecting the gene with pLEX-MCS after digestion, constructing a pLEX-MCS-GADD45a lentivirusplasmid, performing monoclonal sequencing identification and verification, performing cotransfection on the pLEX-MCS-GADD45a lentivirus plasmid and pSPAX.2 and pMD2.G into 293T cells by using a lipofection transfection method, so as to obtain a recombinant lentivirus; and transfecting an IPEC-J2 cell by using the recombinant lentivirus obtained in the step 2), so as to obtain an IPEC-J2 cell linefor highly expressing the pig GADD45a gene. The novel application of the pig GADD45a gene refers to regulation and control on nutrition absorption and metabolism. The invention exploits novel functions of the pig GADD45a gene and novel methods for research the pig GADD45a gene in epithelial cells of intestinal tracts.

The invention relates to construction of a neural tube defect cell model and a cell bank of neural tube defect cell by importing an SV40T gene. The construction is mainly characterized by comprising the following steps: constructing an SV40LTag-pcDNA3.1(I) recon by T4DNA ligase, BamHI, pcDNA3.1(I)DNA and V40LTag DNA; purifying the recon by competent state escherichia coli; importing the recon into in-vitro passaged or logarithmically grown neural tube defect tissue cell digested by collagenase II through a lipofection transfection method to ensure that the recon is integrated with the NDA of the cell, carrying out enlarge cultivation on the G418 screened cell containing a positive recon to clone, and screening a cell of which the cellular morphology, growth curve, karyotype, soft agar colony growth, nude mouse tumorigenesis experiment, SV40 large T gene detection in transfection cell DNA, mRNA expression product measurement and DNA sequence measurement result accord with the immortalized cell characteristics and are same or similar to those of a primary cell as the SV40T mediated neural tube defect in-vitro study cell model which is cryopreserved in liquid nitrogen, so as to lay a foundation for the long-term research of the pathogenesis in vitro from the cell level.

The present invention relates to a method for RNA tagging and analysis on single cell, suitable for platelets and cells with a short half-life, comprising the following steps: a) Providing a population of cells of interest; b) Incubating said cells for a time of 10 minutes to 3 hours, or 30 minutes to 2 hours, at a temperature from about 20° C. to about 37° C., in a culture medium supplemented with a lipofection reagent and a SmartFlare™ probe of interest; c) Fixing with a fixative; d) Visualizing and analysing the RNA of interest.

The invention relates to the technical field of biology, in particular to a recombinant rhabditiform plasmid and application thereof to expression of PCV3 Cap protein and a vaccine. A PCV3 Cap gene iscorrectly inserted into a baculovirus vector, a pFBD-P2C plasmid is constructed, a shuttle plasmid is obtained, the shuttle plasmid is transferred into SF9 insect cells by a lipofection method, recombinant baculovirus is obtained, the PCV3 Cap protein is successfully expressed by an insect expression system, and the molecular weight of the expressed protein is about 23 KDa; and indirect immunofluorescence tests and Western Blot show that Cap protein can be expressed correctly by a baculovirus expression system, and the Cap protein has good antigenicity.

The invention relates to construction of an hTERT-mediated Turner syndrome cell model and a cell bank of Turner syndrome cell by virtue of importing of hTERT, belonging to the medical field. The construction is mainly characterized in that a plasmid pCIneo-hTERT and a vector pLXSNneo are subjected to double enzyme digestion of incision enzymes EcoR I and Xho I, and hTERT and pLXSNneo enzyme-digested products subjected to PCR amplification and gel electrophoresis separation are linked by virtue of Ligation Mix, so as to construct a pLXSNneo-hTERT recombinant; a DH5a competent cell is converted for purification and amplification and an ampicillin-resistant colony is picked to extract the plasmid; the Turner syndrome cell in logarithmic growth undergoes in-vitro passage by virtue of lipofection transfection; the recombinant is integrated with cell DNA, enlarged cultivation is carried out on a cell which is screened out by virtue of G418 and contains a positive recombinant and the cell is cloned; a cell which is consistent with the properties of an immortalized cell in cellular morphology, growth curve, karyotype, nude mice carcinogenicity test, transfection cell telomerase activity, hTERT mRNA expression, immunohistochemistry, cell generation cycle and apoptosis rate and is same or similar to a primary cell is screened out as an hTERT-mediated Turner syndrome cell, and is cryopreserved in liquid nitrogen, so as to lay a foundation for long-term in vitro research on pathogenic mechanism from the view of cellular level.

The invention relates to construction a megakaryocyte model and a cell bank of megakaryocyte by importing of an hTERT recon in the field of medicine. The method is mainly characterized by comprising the following steps: carrying out double enzyme digestion on a plasmid pCIneo-hTERT and a carrier pLXSNneo by incision enzyme EcoR I and Xho I; connecting a PCR amplified and gel electrophoresis separated hTERT and pLXSNneo enzyme-digested product by Ligation Mix; constructing a pLXSNnei-hTERT recon; converting a DH5a competent cell to purify, amplify and pick an ammonium-tolerant benzyl penicillin colony to extract a plasmid, carrying out in-vitro passaging on the logarithmically grown megakaryocyte by lipofection transfection to ensure that the recon is integrated with the DNA of the cell; carrying out enlarge cultivation on the G418 screened cell containing a positive recon to clone, and screening a cell of which the cellular morphology, growth curve, karyotype, nude mouse tumorigenesis experiment, transfection cell telomerase activity, hTERT mRNA expression, immunohistochemical, cell generation cycle and apoptosis rate accord with the immortalized cell characteristics and are same or similar to those of a primary cell as an hTERT-mediated in-vitro study megakaryocyte model which is cryopreserved in liquid nitrogen, so as to lay a foundation for the long-term research of the pathogenesis of related diseases in vitro from the cell level.

The invention relates to construction of a chromosome translocation cell model and a cell bank thereof by virtue of transfection of monkey kidney virus 40 capital T genes, belonging to the filed of the field of medical researches. The construction is mainly characterized in that generally, T4DNA ligase, BamHI, pcDNA3.1 (-) DNA and SV40LTag DNA are used for constructing an SV40LTag-pcDNA3.1 (-) recombinant; the recombinant is purified by competent colon bacillus; and then is introduced to in-vitro passed or logarithmic-growing chromosome translocation tissue cell which is digested by collagenase II by using a lipofection transfection method, so that the recombinant and the DNA of the cell are integrated; cloned cells which are screened by G418 and contain positive recombinants are subjected to enlarging cultivation; the form, growth curve, and karyotype of the screened cells, growth of soft agar colonies, tumorigenesis test of a naked mouse, SV40 capital T gene detection in DNA of the transfected cells, mRNA expression product assay and DNA sequence determination results satisfy the characteristics of immortalized cells; the cells which are the same as or similar to primary cells are used as the SV40LT mediated chromosome translocation in-vitro study cell models and cryopreserved in liquid nitrogen, so that a foundation is laid for researching the pathogenesis of the chromosome translocation in vitro based on the cellular level for a long time.

The invention relates to a T-lymphocytes model and a cell bank thereof established by recombination of SV40L and hTERT gens for medical field. The T-lymphocytes model is characterized in that T4DNA is connected with products passing through BamHI digestion plasmid SV40LTag DNA and vector pc DNA 3.1 (I) DNA to construct an SV40LTag-pcDNA3.1 (I) recombinant; Ligation Mix is connected with products through Eco RI and Xho I double restriction plasmid pCIneo-hTERT and vector pLXSNneo to construct pLXSNneo-hTERT recombinant; the two recombinants are lipofection transfection T-lymphocytes after being purified and identified by competence cell expansion; the cells of positive recombinants screened by G418 are enlarged and cultured to screen cells of which a cellular morphology, a growth curve, a karyotype, a nude mouse carcinogenicity test, a transfection cell telomerase activity, SV40LT and Htert mRNA expression, immunohistochemical, a cell generation cycle and an apoptosis rate conform to the features of immortalized cells andare same or similar to those of primary cells to be used as T-lymphocytes model established by SV40LT and hTERT gene recombination to be stored in liquid nitrogen; the T-lymphocytes model established by recombination of SV40L and hTERT gens lays foundation for in-vitro long-time research of the pathogenesis from cellular level.