274 results about "Co transfection" patented technology

The invention belongs to the technical field of molecular biology and biomedicine, and relates to a method for increasing the efficiency of CRIPSR/Cas9 target knock-out genes NHEJ. According to the method for increasing the efficiency of CRIPSR/Cas9 target knock-out gene NHEJ, small molecule interference RNA that efficiently interferes with expression of PTEN genes is screened out; by using CRISPR/Cas9 system to target knockout genes, the small molecule RNA is cotransfected into the target gene, thus the NHEJ efficiency of target genes is effectively improved and the NHEJ of target gene in cell multiple targets is significantly improved. The method has the advantages of low cost, simple operation and high efficiency, and has a good promoting effect on the efficiency and application of CRISPR/Cas9 technology.

The invention discloses a method for obtaining sheep with different hair colors on the basis of CRISPR/Cas9 and sgRNA of a targeted ASIP gene. The invention provides sgRNA (ASIP-sgRNA) capable of achieving specific and targeted modification of a sheep ASIP gene, and the sgRNA (ASIP-sgRNA) is RNA as shown from the third nucleotide to the 22<nd> nucleotide of 5' tail end of a sequence 4 of a sequence list or RNA with the nucleotides from the third nucleotide to the 22<nd> nucleotide of the 5' tail end of the sequence 4 of the sequence list. The ASIP-sgRNA particularly can be the RNA as shown in the sequence 4 of the sequence list. The invention further provides a method for obtaining sheep with changed hair colors. The method comprises the following steps that co-transfection of sgRNA capable of achieving specific and targeted modification of the sheep ASIP gene and Cas9mRNA on sheep cells is conducted, and therefore the sheep ASIP gene is deleted, and the sheep with the changed hair colors are obtained. According to the method for obtaining the sheep with different hair colors on the basis of CRISPR/Cas9 and sgRNA of the targeted ASIP gene, a CRISPR/Cas9 genome-editing technology is combined with a microinjection technology, and an effective technological means is provided for artificially changing the hair colors of the sheep.

The invention discloses a method for detecting promoter activity by utilizing double luciferase reporter genes. The method for detecting the promoter activity by utilizing the double luciferase reporter genes comprises step 1, building a pGL3-basic-MSTNpro recombinant which contains 7 sections of MSTN gene 5' control region fragments with different lengths; step 2, performing cultivation and planking on target cells, configuring a mixture of the pGL3-basic-MSTNpro of the step 1 and a lipidosome and enabling a renilla luciferase carrier pRL-TK to be served as an internal reference to perform cell co-transfection; step 3, performing detection on luciferase activity through the double luciferase reporter genes.

The invention relates to a preparation method of a CAR-T cell targeting B7H3. The preparation method includes first preparing a PBMC cell; then co-transfecting a 293T cell with a shuttle plasmid LV-B7H3 containing the CAR structure, a helper plasmid psPAX2 and an envelope plasmid VSV-G to obtain a packaged B7H3-CAR virus; then taking a PBMC cell, using anti-human CD3 and anti-human CD28 as activators, culturing and activating for 48 hours and adding the B7H3-CAR virus for infection. By means of the preparation scheme, the expression of IFN-gamma in the CAR-T cell is increased, and the cell killing activity is high. The CAR-T cell targeting B7H3 has a killing effect on various solid tumor cells, has high killing activity, is safe and effective, and can be used for immunotherapy of kidney cancer, lung cancer, liver cancer, glioma, ovarian cancer, breast cancer and the like.

The invention discloses a method for constructing fixed-point integrated exogenous DNA transgenic pigs. The method comprises the following steps: S1, performing safety target screening and target binding gRNA cleavage efficiency verification; S2, constructing a homologous arm donor plasmid and obtaining a fixed-point integrated transgenic cell line; S3, constructing the fixed-point integrated exogenous DNA transgenic pigs. The method disclosed by the invention has the benefits that a gRNA target sequence is introduced in the donor plasmid, the donor plasmid is linearized while a Cas9 nucleasecleavage target gene is induced by utilizing intracellular transcription gRNA, the test steps are significantly simplified, the labor is saved, and the co-transfection efficiency is favorably improved; carriers used for the fixed-point integration are less, a homologous arm is moderate in size, and the transgenic cell line is more favorably obtained; an efficient site-directed integration technology, a high-activity site-specific transgenic cell culture technology and a somatic cell cloning technology are combined, so that the efficient preparation of fixed-point integrated transgenic animalsis facilitated, and the breeding speed of new transgenic animal varieties is accelerated.

The invention provides a preparation method of a recombined new castle disease virus vaccine strain which expresses African swine fever virus p72 proteins and a recombined new castle disease virus vaccine strain. The method provided by the invention comprises the following steps: constructing a recombinant transcriptional plasmid which is inserted with a p72 gene of African swine fever virus (ASFV); constructing a transcriptional helper plasmid system; carrying out a contransfection for the transcriptional plasmids and the transcriptional helper plasmids into host cells BHK-21 which can be duplicated in new castle disease attenuate strains; and saving and obtaining the recombined virus stain. The vaccine strain for expressing the African swine fever virus p72 proteins provided by the invention has important preservation and strategy meaning in the aspect of animal epidemic disease prevention and control, and can be applied to the treatment and prevention of African swine fever virus.

The invention relates to an allele double knockout targeting vector system and a construction method thereof. The system of the invention consists of two complementary vectors, namely pGT-V1 and pGT-V2, wherein each vector contains two in phase LoxP elements which contain a positive selection marker gene Neo/GFP and Hyg/RFP respectively; and the outside of each vector contains a negative selection marker gene TK. Meanwhile, two 8-basic group multiple cloning sites are designed and arranged between the two LoxP elements and the negative selection marker for the insertion of the homology arm. By adopting the constructed targeting vector system of the invention, two complementary targeting vectors are cotransfected into the recipient cell; through the selection of drug and fluorescent double-selection marker, the genetic modification or knockout of the two alleles of the target gene can be realized once; and the interaction of the transfected Cre enzyme and the LoxP elements can be utilized to remove the selection marker gene integrated with the genome, the time of obtaining the homozygous knockout target cell can be shortened, the safety of the transgenic animal can be increased and a valuable technology platform is provided to develop the animal transgenic researches.

The invention provides a construction method of a SOD (superoxide dismutase) gene modified mesenchymal stem cell. The construction method comprises the following steps: (A) connecting an SOD3 gene to an adenovirus vector to obtain a recombinant adenovirus vector; B) co-transfecting the recombinant adenovirus vector and a packaging plasmid to a 293 cell line by utilizing a transfecting agent, culturing until the 293 cell line completely generates a cytopathic effect, collecting an adenovirus strain, storing the strain and confirming that the SOD3 gene is connected to the adenovirus vector; C) amplifying the adenovirus strain, infecting the 293 cell line, collecting the adenovirus supernatant, and determining the titer after ultrafiltration, purification and concentration; and D) infecting the mesenchymal stem cell in different conditions respectively by the adenovirus, and determining the best infection condition to obtain the SOD gene modified mesenchymal stem cell. The EC-SOD (extracellular superoxide dismutase) gene over-expressed SOD gene modified mesenchymal stem cell can be prepared by the method, and can be applied to treatment of mouse with radiation so as to prolong the survival period of the radiated mouse.

The invention relates to an integration plasmid and a cell line expressing recombinant human FVIII, and a construction method and an application thereof. The integration plasmid comprises a rAAV plasmid backbone and a B-zone-deleted FVIII gene connected between two ITR elements in the rAAV plasmid backbone. Further, the integration plasmid can comprise a GFP gene used in subsequent cell line screening and marking, and/or an AAVp5 promoter used for improving integrating efficiency. The cell line comprises HEK293 cells which are obtained by co-transfection by using the above integration plasmid or an integration plasmid constructed with the method above, and plasmid used for expressing AAVRep protein. The HEK293 cells can stably express BDD-FVIII. The cell line can be applied in producing human recombinant coagulation factor VIII. The method provided by the invention has the advantages that: (1) recombinant virus construction and purification processes are avoided; the method is simpler and easier to operate; integration rate is high; recombinant viral genome molecular weight limit is avoided, such that the method can be more widely applied; (2) human HEK293 cell is adopted as a host, and structure and function of expressed FVIII protein are more close to natural FVIII protein.

The invention discloses a screening report vector and screening method for enriching CRISPR/Cas9-mediated homologous recombination repairing cells. The screening report vector includes a universal sgRNA expression cassette, a Cas9 expression cassette, a resistance gene expression cassette and a resistance gene homologous recombination repair template, and the screening reporter vector itself can be repaired by homologous recombination of the resistance gene sequence to make the cells have resistance. The screening report vector, a targeting vector for cell genome HDR editing and a donor vectorform a co-transfection system which can be applied to HDR positive cell cloning with efficient enrichment point editing, fragment insertion and fragment deletion, and can be widely applied to a variety of cells of mammals. The screening efficiency of HDR-edited cells is significantly improved, the reintroduction of double-strand breaking and DNA integration in the unrelated positions of the cellgenome is not required, and an effective way is provided to promote research and application of accurate gene editing.

The invention relates to the technical field of biology, in particular to a leukemia mouse model based on a gene co-transfection technology and a preparation method thereof. The preparation method of the leukemia mouse model comprises the following steps: performing construction and packaging of K-ras mutants and AML1-ETO fusion gene lentivirus vectors; performing bone marrow cell separation and virus infection condition monitoring; implanting infection cells in a mouse to build a leukemia animal model; and performing model identification. The method of building the leukemia mouse model in a mode of utilizing caudal vein injection to lead in the manual site-directed mutagenesis K-ras mutants and AML1-ETO fusion gene lentivirus vectors is adopted initiatively. The leukemia mouse model is high in success rate, pathological characteristics are high in similarity to morbidity conditions of clinical leukemia, and the novel animal model can be provided for leukemia extramedullary infiltration mechanism research, leukemia medicine screening and gene and molecule target treatment.

The invention discloses a cell line for stable expression of a peste des petits ruminants virus receptor Nectin-4 and a construction method of the cell line. According to the invention, by virtue of asynthetic Nectin-4 gene and on the basis of a lentiviral vector system, a recombinant plasmid pLOV-eGFP-Nectin-4 is constructed. The recombinant plasmid, together with pSPAX2 and pMD2.G plasmid, is applied to co-transfection of a 293T cell, so that retrovirus-like particles are obtained. A supernatant-infected MDBK cell is cultivated by virtue of cells containing the retrovirus-like particles, and puromycin screening and purifying are conducted, so that the MDBK cell for stable expression of Nectin-4 protein is obtained, and the MDBK cell is named as MDBK-Nectin-4. Through the construction ofthe cell line, an experimental material is provided for deep research of an interaction between PPRV and the receptor Nectin-4 and for the clinical rapid isolation of the PPRV is provided.

The invention discloses a Cre fusion protein functional test cell and a modified Cre fusion protein. The Cre fusion protein functional test cell takes an HEK (human embryonic kidney) 293 cell as a host cell, a pAcmy (African cassava mosaic virus)-stop-EGFP (enhanced green fluorescent protein) carrier and phi C31 integrase are used for co-transfection of the host cell. Through the action of the phi C31 integrase, the carrier which carries an LoxP (locus of X-over P1) element having a Cre recombinase detection function is integrated to a pseudo attP (attachment P) site of an HEK 293 cell line in a single copying manner, so a stable and reliable cell model is provided for researching the Cre fusion protein membrane penetrating efficiency and biological activity. The Cre fusion protein HNTC (hemagglutinin-neuraminidase cytotoxic T) which is modified by TAT (transactivator) and NLS (nuclear localization signal) polypeptides has high membrane penetrating efficiency and good biological activity, can be effectively used as a tool for genetic manipulation at a mammalian cell chromosome level.

The invention clones insect specific neurotoxin gene BmkIT of East Asian scorpion and insect chitinase gene Chi to an insect cell-converting medium carrier pAcPDIEI to obtain a recombinant plasmid containing divalent anti-insect genes pAcPDIEI-Bmkit-Chi and transfecting the recombinant plasmid and DNA of AcNPV genome together in sfg cells, and obtaining a recombinant rhabdovirus containing divalent anti-insect genes AcNPV-BmKIT-Chi. The cooperation of the insect specific neurotoxin with the chitinase improves the poisoning effect of virus on cotton bollworms and other lepidopteral pests.

The invention provides a COVID-19 vaccine and a preparation method and application thereof. The COVID-19 vaccine comprises T cells expressing SARS-CoV-2 S protein. The preparation method of the COVID-19 vaccine comprises the following steps that (1), coding genes of the SARS-CoV-2 S protein are inserted into an adenovirus vector to construct a recombinant adenovirus vector; (2), the recombinant adenovirus vector and packaging helper plasmids are used for co-transfecting mammalian cells to prepare recombinant adenovirus; and (3), the recombinant adenovirus is adopted for infecting the T cells to obtain the COVID-19 vaccine. According to the COVID-19 vaccine and the preparation method and application thereof, a virus system is utilized for constructing the recombinant T cells expressing theSARS-CoV-2 S protein, the recombinant T cells are input into a body to continuously express the S protein, the body is induced to generate a specific humoral immune response or a cellular immune response, and the body obtains the protective immunity capacity on SARS-CoV-2.

The invention discloses a preparation method of a novel coronavirus pneumonia bivalent vaccine. The preparation method comprises the following steps: amplifying shRNA of a 2019-nCoV targeted interfering gene, performing enzyme digestion on the PCR product, constructing an interfering vector pSilencer-shRNA, performing DH5a transformation, constructing a shuttle vector pDC312-shRNA, co-transfectingHEK293 by the shuttle vector pDC312-shRNA and adenovirus framework plasmid pBHGloxAEl to prepare Ad-shRNA, and then preparing Ad-nCoVdsRNA. The preparation method further comprises the following steps: amplifying a 2019-nCoV antibody expression gene, performing enzyme digestion on the PCR product, constructing a shuttle plasmid pShuttle-nCoV, performing DH5a transformation, performing PCR amplification, co-transfecting HEK293 by the amplified shuttle plasmid pShuttle-nCoV and adenovirus skeleton plasmid pAd-nCoV to prepare a recombinant adenovirus Ad-nCoV, and further preparing Ad-nCoVDNA, and then preparing the bivalent vaccine by using the Ad-nCoVds RNA, the Ad-nCoVDNA and H20 according to a ratio of 1:1:19. After spray inoculation through a respiratory tract, the adenovirus (Ad) introduces nCoVds RNA and nCoVDNA into the cells.

The present invention provides a method for preparing and screening a cell line expressing bispecific antibody. The method comprises: constructing two plasmids expressing bispecific antibody, wherein the two plasmids contain double promoters and respectively express different fluorescent proteins; and carrying out transformation, culture and extraction on the two recombinant plasmids, co-transfecting the two recombinant plasmids into host cells, screening the positive monoclonal cell line expressing the double fluorescence, and evaluating the yield and the stability according to the fluorescence intensity. With the method of the present invention, the high yield cell line can be conveniently and rapidly screened in advance, the test period can be shortened, the manpower and material resource investment can be reduced, and the accuracy is high.

The invention discloses a recoverable immortalized rat bone marrow mesenchyme stem cell. The cell carries an SV40T (Simian Virus 40T) gene and a hygromycin resistance gene and two ends of the SV40T gene also have orthokinetic Loxp sites. The preparation method of the recoverable immortalized rat bone marrow mesenchyme stem cell comprises the following steps of: co-transfecting an HEK293 (Human Embryo Kidney 293) cell by SSR69 (Simple Sequence Repeat 69) plasmids and pAmhpo plasmids and screening by the hygromycin to obtain the recoverable immortalized rat bone marrow mesenchyme stem cell with hygromycin resistance; the cell has the same biological property with a primary rat and also has the advantages of strong external multiplication capacity, stable biological property, capability of being recovered to a previous state, good biological safety and the like; and the cell provided by the invention can be used as a seed cell induced and differentiated by nerves and a seed cell for treating hypoxicischemic brain damage of a newly-born rat.

The invention belongs to the technical field of gene engineering and particularly relates to a method for restraining mesenchymal stem cells from differentiating into fat cells. A purpose of restraining the mesenchymal stem cells from differentiating into the fat cells is realized by the following steps of: constructing recombinant retrovirus plasmids containing ICAM-1 (intercellular cell adhesion molecule) genes, carrying out co-transfection on the packaging cells by the recombinant retrovirus plasmids together with packaging plasmids, collecting relevant viral supernatants, and infecting the mesenchymal stem cells. According to the method for restraining the mesenchymal stem cells from differentiating into the fat cells, the separation method is simple in operation, convenient and practical, the efficiency (more than 90% of ICAM-1 can be highly expressed) for transfecting the mesenchymal stem cells is high, and the obtained mesenchymal stem cells are exuberant in in-vitro multiplication and can be passed down for multiple times (more than 50 generations). Thus, as the method and a culture technique system for restraining mesenchymal stem cells from differentiating into fat cells are established, a foundation for research and application of the mesenchymal stem cells is laid.

The invention discloses a recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, a recombinant adenovirus and a construction method. The recombinant adenoviruscan synchronously express the African swine fever p72 and B602L proteins after infecting cells, and the B602L is serially expressed with the p72 through self-splitting decomposition 2A signal peptide.According to the invention, a recombinant adenovirus vector capable of simultaneously expressing p72 and B602L proteins is constructed; the recombinant adenovirus capable of expressing African swinefever virus p72 and B602L proteins is obtained after cells are co-transfected by using the vector and an adenovirus skeleton system, a test animal can be stimulated to generate a specific antibody after the recombinant adenovirus is used for immunizing the test animal, and new test data is provided for the development of African swine fever vaccines.