281 results about "Co transfection" patented technology

The invention discloses a CRISPR / Cas9-gRNA targeting sequence pair, a plasmid and an HD model of HTT, and relates to the field of biotechnology. The targeting sequence pair of the invention comprisesan L sequence and an R sequence. The base sequence of the L sequence is shown as SEQ ID NO.1. The base sequence of the R sequence is shown as SEQ ID NO.2. The targeting plasmid of the invention comprises a first vector plasmid and the above-mentioned targeting sequence pair. The targeting sequence pair is constructed into the first vector plasmid. The HD cell model of the invention is obtained byco-transfecting cells with PolyQ Donor plasmids and the above-mentioned targeting plasmids. By using differentiated neuron cells as a carrier to construct the HD cell model, the invention provides a research platform for exploring the influences or changes of mHtt protein on the internal environment of differentiated nerve cells, and studying the changes of various signal pathways, metabolic pathways and intracellular homeostasis caused by mHtt protein in differentiated neuronal cells.

IgM can be obtained in the form of a pentamer by placing the genes encoding the H, L, and J chains on the same vector to transform appropriate host cells. The gene encoding the J chain may be introduced by co-transfection. When no J chain is expressed, the IgM is produced as a hexamer. The transformants obtained according to the present invention achieve a high yield of IgM. The present invention also provides methods which enable separation and quantification of polymeric IgM.

A recombinant adeno-associated virus (rAAV) expressing antisense human CYP2J2 gene produced by a method of co-transfection into 293 cell by calcium phosphate precipitation, the rAAV having the following three plasmids: a) pXX2: a packing plasmid having a nucleotide sequence encoding AAV Rep and Cap protein, wherein the nucleotide sequence is operably linked upstream and downstream to a p5 promoter respectively to increase expression 15-fold; b) pXXUF1-anti2J2: an eukaryotic expression vector having a CMV promoter, a NotI site for insertion of target genes, the CYP2J2 gene oriented in the 3′ to 5′ direction, and inverted terminal repeats (ITRs); and c) pXX6: a helper plasmid deleted of the pathogenic gene sequences of adenovirus and having an E1A, an E2A, and a VA1 RNA gene of adenovirus. Also provided is a method of preparing the rAAV.

The invention belongs to the field of micro-organism animal cell line and relates to a human hepatoma cell line which can emit high-intensity red or green fluorescence and has high transferring ability of lung and lymph node metastasis, and a method for establishing the same. The method comprises the following steps: using the human hepatoma cell line HCCLM3 and HCCLM6 which have high transferring ability of the lung and lymph node metastasis as mother cells, performing cotransfection on plasmid DNA of 239 cells through slow virus packaging plasmids to obtain false slow virus particles by expressing red or green fluorescent protein genes through eucaryon, and infecting liver cancer cell strains of the mother cells to obtain the chromosome integrated hepatoma cell line which has high transferring ability of the lung and lymph node metastasis and can stably expressing the red or the green fluorescence. The human hepatoma cell line which has high transferring ability of the lung and lymph node metastasis in vitro can be applied to the tracer studies on tumor cells, the molecular mechanism studies on the recurrence and transferring of liver cancer, as well as the pre-clinical drug efficacy studies on new anti-tumor drugs, thus the human hepatoma cell line has wide application prospect.

IgM can be obtained in the form of a pentamer by placing the genes encoding the H, L, and J chains on the same vector to transform appropriate host cells. The gene encoding the J chain may be introduced by co-transfection. When no J chain is expressed, the IgM is produced as a hexamer. The transformants obtained according to the present invention achieve a high yield of IgM. The present invention also provides methods which enable separation and quantification of polymeric IgM.

The invention discloses a preparation method of bone mesenchymal stem cells carrying NK4 genes and application thereof in medicine for treating gastric cancer. The preparation method comprises the following steps: extracting the NK4 genes from an HGF plasmid, using an in-Fusion technology to directionally clone the NK4 genes to a slow virus expression carrier plasmid, constructing an NK4-EGFP fusion gene recombinant slow virus carrier plasmid (pGC-FU-NK4) and then co-transfecting a 293T cell with the pGC-FU-NK4, a pHelper1.0 carrier and a pHelper2.0 carrier, packaging to produce NK4 overexpression slow virus particles (Lenti-NK4), using slow virus (Lenti-NK4) carrying NK4-EGFP fusion gene to transfect the bone mesenchymal stem cells to establish in vitro bone mesenchymal stem cells to stably express the NK4 genes. Balb / C nude mice animal experiments prove that the NK4 gene therapy taking BMSCs as a carrier can obviously inhibit the growth of subcutaneous transplant tumor of gastric cancer with high tumor inhibition efficiency, and the NK4 gene therapy is a good gene therapeutic method for gastric cancer.

A high-activity glutathion peroxidase GPX 1 mutant and its preparation method belong to the field of biotechnology. A mutant gene is obtained by a gene mutation or synthesis method, and then a catalytic group SeCys of GPX is introduced into a substrate binding part of the mutant through an auxotroph prokaryotic expression system or an auxotroph and SPP low-temperature combined expression system so as to endow the mutant with high GPX activity; or a target gene, along with a SeCys insertion sequence, is firstly assembled onto a secreting type mammalia cellular expression vector, and then a binding protein 2 of the SeCys insertion sequence is assembled onto an intracellular mammalia cellular expression vector, contransfection of the same lactation cell strain is carried out by the two vectors, and GPX is synthesized by the lactation cell in the presence of sodium selenite. The method provided by the invention is simple. The mutant provided by the invention has advantages of high activity, high yield and good stability. Yield decreasing and inactivation caused by renaturation of an inclusion body are avoided. Thus, natural GPX source limitation and unstable properties are solved.

The invention discloses a recombinant Newcastle disease virus for expressing goose parvovirus VP3 genes and a construction method thereof, belonging to the field of recombinant virus vaccines. The recombinant Newcastle disease virus is a goose isolate, the cleavage site amino acid sequence of F protein of the recombinant Newcastle disease virus is GRQGRL, and the P3 gene is positioned in a noncoding region between the P gene and M gene of Newcastle disease virus. The transcription plasmid pCI-NA-VP3(SEQ ID NO.1) and the transcription helper plasmid pcDNA-N, pcDNA-P and pcDNA-L(SEQ ID NO. 2-4) cotransfect a host cell licensed by application of the Newcastle disease virus to culture a transfected host cell, and the recombinant Newcastle disease virus can be saved from a cell suspension of the transfected host cell. The recombinant Newcastle disease virus for expressing goose parvovirus VP3 genes can be used as a bivalent living-vector vaccine for preventing Newcastle disease virus and goose parvovirus.

The invention discloses a specific targeted hDGK theta gene luciferase reporting system. The specific targeted hDGK theta gene luciferase reporting system is composed of a targeting carrier and a targeting donor, wherein an exogenous DNA fragment required to be introduced is carried by the targeting donor, the exogenous DNA fragment comprises T2A small peptide, a luciferase gene cDNA sequence, aneGFP expression frame, a Neomycin gene sequence, and an upstream and downstream homologous arm sequence; and the targeting carrier contains a nuclease Cas9 expression frame and a guiding chain sgRNA expression frame of the targeted hDGK theta. An establishing method of the reporting system comprises the following steps: screening sgRNA of a targeted hDGK theta gene 3' non-coding region, constructing an eukaryotic expression carrier carried by Cas9 and a sgRNA expression element as the targeting carrier, constructing the upstream and downstream homologous arms carried with a luciferase gene cDNA sequence and used for targeted integration as well as the targeting donor used for gene screening, performing co-transfection of target cells by two carriers, screening by using a resistant gene, and establishing the luciferase reporting system of targeted hDGK theta in a target cell.