24 results about "Stable gene" patented technology

The invention belongs to the technical field of molecular markers, and particularly relates to a QTL gene for regulating and controlling maize fusarium graminearum stem rot resistance, a KASP molecular marker and application of the QTL gene and the KASP molecular marker. A maize high-disease-resistance inbred line H1710 and a high-susceptibility inbred line Huangzao 4 are used for constructing an F2 group, and high-disease-resistance and high-susceptibility samples are selected from the F2 group for constructing a mixed pool for BSA-seq analysis; in combination with bioinformatics analysis, molecular markers and fine positioning, a stable QTL gene for controlling the resistance of the fusarium graminearum stem rot of the corn is positioned from the sixth chromosome bin6.01 of the corn. On the basis, the invention provides a corn genome region capable of improving the corn stem rot resistance and a corresponding DNA (Deoxyribose Nucleic Acid) fragment thereof. In addition, the invention also provides a nuclear KASP molecular marker linked with the QTL locus. After the molecular marker is screened and verified, the SNP molecular marker provided by the invention can be used for efficiently and quickly identifying the genotype of a to-be-detected corn material and screening a stem rot resistant material.

This invention discloses a kit and method for extracting cell nuclei from plant tissues, belonging to the field of biotechnology. The kit includes a nuclear lysis buffer and a nuclear washing buffer. The nuclear lysis buffer is prepared using nuclease-free water with sucrose, Tris-HCl (pH 7-8), NaCl, MgCl2, a surfactant, DTT, an RNase inhibitor, and spermidine. The nuclear washing buffer is prepared using nuclease-free water with sucrose, Tris-HCl (pH 7-8), NaCl, MgCl2, DTT, an RNase inhibitor, and spermidine. This kit is suitable for extracting cell nuclei from fresh yew stem tissue, yielding cell nuclei with intact nuclear membranes, stable gene expression, high purity, and few impurities, suitable for single-cell sequencing.

The invention belongs to the technical field of breeding planning, and particularly relates to a large model construction method and system for multi-source data collaboration pear precision breeding planning, and the method comprises the steps: obtaining a multi-source collaboration data set, constructing a five-dimensional incidence matrix, and building a unified correlation basis of multi-dimensional data; constructing a constrained knowledge expression structure and training a constraint consistency reasoning model on the basis, analyzing a user breeding instruction in a constraint driving mode, and generating a combined constraint condition set containing a gene feasible region and a multi-phenotype collaborative target; calling germplasm resources under a cross-domain feature alignment and feature-level desensitization framework, executing phenotype-genotype collaborative screening, parent dynamic matching and multi-generation genetic evolution deduction, and generating breeding planning simulation data; and finally, outputting an optimal breeding scheme through a multi-dimensional evaluation system, and continuously updating the model by utilizing verification data. According to the invention, unification of multi-phenotype balanced synergistic improvement and stable gene transfer is realized, and the breeding efficiency and variety adaptability are remarkably improved.

Disclosed herein are prokaryotic and eukaryotic microbes, including E. coli and S. cerevisiae, genetically altered to biosynthesize tryptamine and tryptamine derivatives. The microbes of the disclosure may be engineered to contain plasmids and stable gene integrations containing sufficient genetic information for conversion of an anthranilate or an indole to a tryptamine. The fermentative production of substituted tryptamines in a whole-cell biocatalyst may be useful for cost effective production of these compounds for therapeutic use.

This invention discloses a plasmid for gene overexpression in non-pattern *Bacillus laterosporus*. Its leader region contains a methyltransferase expression cassette derived from the *Bacillus laterosporus* i83 genome, enabling stable existence in specific hosts containing the RM system. Based on this, a PidR2 promoter is added to obtain the pMCPidR2 shuttle plasmid. The PidR2 promoter effectively drives target gene expression, the pWV01 replicon ensures plasmid replication in non-pattern hosts, and the chloramphenicol resistance gene is used for positive colony selection. The plasmid contains a TraJ element, allowing it to be introduced into non-pattern microbial hosts via conjugation transfer. This invention also provides a target gene expression strategy in non-pattern microorganisms, achieving high transformation efficiency and high strain yield through conjugation transfer transformation of donor bacteria, conjugation transfer helper bacteria, and recipient bacteria, resulting in stable expression of the target protein. This invention provides an effective vector for efficient and stable gene overexpression in specific non-pattern microorganisms, helping to solve the problem of difficult genetic manipulation of industrial production strains.

The application provides a method for constructing a Bmp4 gene knock-in mouse model, and the method comprises the following steps: obtaining sgRNA12 shown in SEQ ID NO. 3; mixing the sgRNA12, Cas9 mRNA and a targeting vector shown in SEQ ID NO. 1, and then microinjecting into a mouse zygote to obtain F0 generation mice; selecting F0 generation positive mice in genotype identification results of the F0 generation mice, and mating the F0 generation positive mice with wild type mice to obtain F1 generation mice with stable genotypes; and screening gene targeting mice with correct recombination, and mating the gene targeting mice with retinal specific knock-out tool mice Six3-Cre to obtain Bmp4 gene knock-in model mice. The method has high gene editing efficiency, and the transgenic mouse can highly express human Bmp4 protein.

Provided herein are compositions and methods for improved synthesis-mediated transgene insertion. The methods can include modulation of DNA repair pathways to inhibit stable gene insertion without base-pairing between the genome-side and insertion-side DNA sequences, as useful with other methods based on host cell DNA repair pathway manipulation to improve functional full-length intact transgene synthesis.

In certain aspects, the present invention provides methods for inducing a stable gene modification of a target nucleic acid via homologous recombination in a primary cell, such as a primary blood cell and / or a primary mesenchymal cell. In certain other aspects, the present invention provides methods for enriching a population of genetically modified primary cells having targeted integration at a target nucleic acid. The methods of the present invention rely on the introduction of a DNA nuclease such as a Cas polypeptide and a homologous donor adeno-associated viral (AAV) vector into the primary cell to mediate targeted integration of the target nucleic acid. Also provided herein are methods for preventing or treating a disease in a subject in need thereof by administering to the subject any of the genetically modified primary cells or pharmaceutical compositions described herein to prevent the disease or ameliorate one or more symptoms of the disease.

The invention relates to the technical field of gene engineering technology, in particular to a preparation method of an efficient and stable gene knockout cell line. According to the AKAP11 gene knockout cell line provided by the invention, the 6th exon of the AKAP11 gene in the cell line is subjected to 3395bp frameshift deletion mutation, so that a coding frame is shifted, and protein translation is terminated in advance; after continuous subculture, the homozygous knockout phenotype is still maintained through PCR sequencing verification. A CRISPR / Cas9 system is combined with puromycin screening, the positive cloning rate reaches 30% or above, the construction period is shortened to 45 days, the constructed cell line can be directly used for AKAP11 related cell signal path research, disease model construction and targeted drug screening, and a reliable tool is provided for basic research and clinical transformation.

This invention relates to the field of vaccine technology and provides a CHO cell line for exocrine expression of PEDV GIIb subtype S protein and its application. The CHO cell line integrates a codon-optimized PEDV GIIb S protein encoding gene carrying the TPA signal peptide into its chromosome. The nucleotide sequence of the PEDV GIIb S protein encoding gene is shown in SEQ ID NO.1. This invention uses currently prevalent GIIb strains as the antigen source to ensure vaccine strain matching; it constructs a secretory expression structure, thereby significantly improving the exocrine expression efficiency of the S protein; it precisely truncates the transmembrane region while retaining key structural domains, maintaining the protein's native conformation and immunogenicity; and it obtains a CHO cell line with stable gene integration and long-term passage to maintain expression levels through optimized transfection conditions and an antibiotic selection system.

To provide engineered cells using the cytotoxic activated natural killer cell line as the basis to improve immunotherapies to cancer and tumors.SOLUTION: The present invention provides a recombinant modified natural killer (NK)-92 cell stably transfected with nucleic acids, wherein the recombinant modified NK-92 cells have American Type Culture Collection (ATCC) deposit number CRL-2407; the nucleic acid encodes an anti-cluster of differentiation 123 (CD123) chimeric antigen receptor (CAR), an Fc receptor, and a cytokine; and the recombinant modified cells express the anti-CD123 CAR and the Fc receptor on the cell surface of the recombinant cells.SELECTED DRAWING: Figure 16

The invention discloses a kit for extracting plant tissue cell nucleuses, and belongs to the technical field of biology. The kit comprises a cell nucleus lysis solution and a cell nucleus cleaning solution, the cell nucleus lysis solution is prepared from cane sugar, Tris-HCl with the pH value of 7-8, NaCl, MgCl2, a surfactant, DTT, RNase inhibitor and spermidine by using nuclease-free water, and the cell nucleus cleaning solution is prepared from sucrose, Tris-HCl with the pH value of 7-8, NaCl, MgCl2, a surfactant, DTT, RNase inhibitor and spermidine by using nuclease-free water. The cell nucleus cleaning solution is prepared from sucrose, Tris-HCl, NaCl, MgCl2, DTT (dithiothreitol), RNase inhibitor and spermidine by using nuclease-free water, wherein the pH (Potential of Hydrogen) of the Tris-HCl is 7-8. The kit is suitable for cell nucleus extraction of fresh tissues of taxus chinensis stems, the obtained cell nucleus is complete in nuclear membrane, stable in gene expression, high in purity and few in impurity, and the kit can be used for single cell sequencing.

The invention belongs to the technical field of plant stem cell culture, and particularly relates to an isolation and differentiation culture method of salvia miltiorrhiza root cambium stem cells, which comprises the following steps: cutting the root of a wild salvia miltiorrhiza plant into sections, washing and sterilizing, cutting into small sections, sterilizing for the second time, and washing to obtain a sterile explant. Or carrying out pretreatment, surface sterilization and culture on salvia miltiorrhiza seeds to obtain sterile seedlings, carrying out expanding propagation and rooting and seedling strengthening culture to obtain sterile plants, and cutting the roots of the sterile plants into sections to obtain the sterile explants. And soaking the sterile explant in a preserving fluid, and then carrying out induction culture, subculture and suspension culture to obtain the salvia miltiorrhiza root cambium stem cells. Wherein the preserving fluid, the induction culture medium, the subculture medium and the suspension culture medium all contain plant hormones. And the induction culture medium also contains REF1. According to the separation and differentiation culture method of the salvia miltiorrhiza root cambium stem cells, cell browning can be effectively prevented, and the cultured tissue cells are stable in gene expression and growth.

The invention discloses a method for constructing a thrombocytopenic purpura animal model based on a C57BL / 6J background mouse, and belongs to the technical field of genetic engineering, and the method comprises the steps of carrier construction, preparation and in-vitro transcription, fertilized egg microinjection, mouse identification, mating and breeding, drug induction and the like. According to the method, the limitation of the genetic background and strain of mice is broken through, and the model is directly constructed on the widely used C57BL / 6J background, so that TTP related experiments can be more accurately carried out, other genetic factors, especially uncertain influences caused by the complex genetic background, are eliminated, errors of the experiments are avoided, and the experimental accuracy is improved. The method disclosed by the invention has the advantages of simplicity and convenience in operation, high targeting precision, greatly shortened knockout period and the like; through efficient and stable gene editing, the animal model which is high in survival rate and remarkable in pathological expression is obtained, the continuous simulation capacity of the disease course is supported, and a disease model is provided for TTP pathogenesis, drug screening and treatment strategy optimization.

The invention provides a lung targeting lipid nanoparticle (LNP) based on cationic lipopeptide as well as a preparation method and application of the lung targeting lipid nanoparticle. The lung targeting LNP is composed of cationic lipopeptide, DLin MC3-DMA, cholesterol and DMG-PEG 2000, and nucleic acid can be efficiently encapsulated and compressed through electrostatic interaction, so that high nucleic acid encapsulation efficiency is obtained. Based on the positive charges carried by the cationic lipopeptide, the LNP can adsorb specific biomolecules in plasma to form a protein crown structure, and the lung cell endocytosis of the LNP is promoted by utilizing the corona effect, so that efficient active targeting on the lung is realized. The LNP provided by the invention can specifically deliver nucleic acid to the lung, supports stable gene expression, and is suitable for treating related diseases of the lung. Compared with an existing SORT LNP technology, a large amount of cationic lipopeptide does not need to be additionally added, the total lipid dosage is low, and better biocompatibility and safety are shown.

The invention belongs to the technical field of biology, and particularly relates to a mouse model for systemic expression or tissue specific expression of KRAS-G12D-EGFP fusion protein and a preparation method thereof, and the preparation method comprises the following steps: a) screening gRNA target sites; b) constructing a homologous recombinant vector; c) mixing the homologous recombinant vector, gRNA and Cas9 mRNA, injecting the mixture into a mouse fertilized egg, and transplanting the mixture into a mouse blastocyst; d) carrying out PCR (Polymerase Chain Reaction) screening on the F0-generation mice to obtain correct homologous recombination positive mice; and e) mating the positive F0-generation mouse with a wild type mouse, breeding, and carrying out PCR screening to obtain an F1-generation recombinant positive mouse. A mouse model modified by the gene is stable in heredity and can be used for researching the biological function of the KRAS-G12D mutant protein in a mouse body. Particularly, the mouse model can be used for researching the function of the KRAS-G12D mutant protein at the tissue specificity level, and the mutant protein can be subjected to cell tissue tracing by using EGFP fluorescent protein, so that the mouse model has a certain application value.

The application belongs to the field of crop genetic breeding, and relates to application of a positive regulation factor ZmGI2 in breeding drought-resistant maize varieties. ZmGI2 belongs to the GIGANTEA family protein, is located in the nucleus, has transcription activation activity, and the expression of the gene can be significantly induced by drought stress. The antioxidant enzyme activity of a ZmGI2 overexpression strain is improved, the contents of malondialdehyde and hydrogen peroxide are reduced, and the water retention capacity of leaves is enhanced. In the reproductive growth period, ZmGI2 can alleviate the inhibitory effect of drought on the development of female and male ears, shorten the flowering interval of female and male flowers, and greatly reduce the yield loss caused by drought. The application adopts pFGC5941 to construct a ZmGI2 overexpression vector, relies on a mature maize genetic transformation system to create an overexpression strain, the whole set of technical system is mature and stable, and the ZmGI2 gene, the vector and the genetic material can be used for maize drought-resistant germplasm creation, drought-resistant new variety breeding and stable yield cultivation in drought areas.

The invention discloses a novel gene expression plasmid pTAEP2 capable of realizing efficient and stable overexpression in brevibacillus laterosporus (Brevibacillus laterosporus), and a preparation method of the novel gene expression plasmid pTAEP2 capable of realizing efficient and stable overexpression in brevibacillus laterosporus. The plasmid contains a toxin-antitoxin system gene cassette, so that the plasmid can stably exist in a specific non-mode host; the P2 promoter can drive high-level expression of a target gene; the RepF replicon is contained, so that the plasmid can be effectively replicated in a non-mode host; the composition contains ampicillin antibiotics and erythromycin which are used as selection markers. The invention also provides a method for target protein expression by transforming specific non-mode brevibacillus laterosporus with the pTAEP2 plasmid, the plasmid contains a TraJ element, so that the plasmid is efficiently introduced into a non-mode microbial host including brevibacillus laterosporus in a conjugational transfer manner, the gene introduction process is greatly simplified, and the production cost is reduced. And stable and high expression of a target gene is realized. The invention provides a powerful molecular biological tool for developing efficient and stable gene overexpression research and industrial application, and is beneficial to efficient gene manipulation in the non-model bacteria.

The invention discloses a method for performing efficient and stable gene editing on dendrobium officinale based on CRISPR / Cas9 mediation of a GFP (Green Fluorescent Protein) marker. The method for performing efficient and stable gene editing on dendrobium officinale comprises the following steps: designing gRNA (guide Ribonucleic Acid); constructing a CRISPR / Cas9 gene editing vector with a GFP (Green Fluorescent Protein) marker; infecting the callus of the dendrobium officinale by an agrobacterium transformation method; and counting the transformation efficiency and the editing efficiency of the CRISPR / Cas9 gene editing system. Based on a regeneration system of dendrobium officinale callus, GFP is used as a selection marker, and an efficient CRISPR / Cas9 gene editing platform is established in dendrobium officinale. The CRISPR / Cas9 gene editing method can be used for carrying out function research on the related genes of the dendrobium officinale and promoting genetic improvement and molecular breeding of the dendrobium officinale.

This invention provides expression-enhancing nucleotide sequences for eukaryotic expression systems, enabling enhanced and stable expression of recombinant proteins in eukaryotic cells. It also provides genomic integration sites for enhanced expression and methods of use for expressing genes of interest in eukaryotic cells. Furthermore, it provides chromosomal loci, sequences, and vectors for enhanced and stable gene expression in eukaryotic cells.

The invention discloses an NFE2L3-multi-tag fusion stable cell line, a special tool and a construction method and application of the NFE2L3-multi-tag fusion stable cell line, and belongs to the technical field of crossing of molecular biology and cell biology. According to the invention, a CRISPR-Cas9 gene editing technology is adopted, multiple types of tags such as GFP, Flag and the like are inserted in the same frame in front of a termination codon of an NFE2L3 gene, and specific sgRNA targeting NFE2L3, a universal HDR donor vector and a construction composition are provided in a matched manner. The construction method comprises the steps of sgRNA design, donor vector construction, adaptability delivery and positive screening, and overcomes the defects of unstable expression, gene function interference and the like in the prior art. The obtained cell line can avoid the problem of insufficient NFE2L3 special antibodies, is suitable for ChIP-seq, living cell imaging and other experiments, assists downstream target gene analysis, upstream regulatory factor screening and stress condition response identification, and provides an efficient tool for NFE2L3 functional mechanism and tumor related research.

The invention discloses a preparation process and application of a gene stable knock-down cell line, and belongs to the technical field of molecular biology. SMURF2 stably transfected strains are obtained by combining targeted shRNA lentivirus infection with puromycin screening. Experiments prove that SMURF2 knock-down can promote apoptosis of cancer cells and inhibit metastasis, and a new model and a molecular target are provided for targeted therapy of nasopharyngeal carcinoma.