322 results about "Reporter gene" patented technology

The invention discloses a SbWRKY51 gene, a promoter and application of the SbWRKY51 gene and the promoter in improvement of salt tolerance of sorghum, and belongs to the technical field of plant genetic engineering. The SbWRKY51 gene is screened from sorghum, and the expression level of the SbWRKY51 gene is up-regulated after salt stress treatment. The protein coded by the gene belongs to a WRKY transcription factor family, has transcriptional activation activity, and can start the expression of a reporter gene. Sorghum overexpression strains and complementary strains of the gene are obtained, salt tolerance related physiological indexes are measured, overexpression of the gene can improve the seed germination rate, increase the seed root length, regulate ion balance and enhance the free radical scavenging capacity of plants, and the effect of the gene in the aspect of improving the salt tolerance is embodied. A further research finds that the SbWRKY51 gene can enhance the plant salt tolerance by regulating a lignin synthesis pathway. The technical scheme provides a basis for cultivating resistant plants, and has important guiding significance for enhancing the production potential of sorghum under a high-salt condition and promoting agricultural development.

The invention provides a CsERECTA gene-based tea tree leaf shape regulation and control method and a tea tree light utilization rate improving method, which are characterized in that a sequence of a tea tree gene is cloned to obtain a complete sequence; then, the CsERECTA is subjected to bioinformatics analysis, expression of a GUS reporter gene is started by constructing a plant expression vector and connecting a target fragment, and a recombinant plant vector is used for genetic transformation of tobacco, so that the function of the CsERECTA is verified; the character of the CsERECTA transgenic line is analyzed, the leaf shape is determined, the method capable of improving the leaf shape of the tea tree is obtained, and a foundation is laid for cultivating a new variety and improving the light utilization rate of the tea tree by using the CsERECTA gene.

The invention relates to the technical field of medicine screening, in particular to an active substance screening platform and application thereof. The active substance screening platform comprises a cell combination; the cell combination comprises immunoregulation screening cells and antioxidant screening cells; the immunoregulation screening cell is a mouse mononuclear macrophage RAW 264.7 carrying an NF-kappa B element conserved sequence and a reporter gene; the antioxidant screening cell is a porcine small intestine epithelial cell IPEC-J2 cell carrying an ARE element conserved sequence and a reporter gene. The invention provides a screening platform based on a specific cell combination, which can quickly obtain an active substance with immune regulation, antioxidation or two functions from a large number of candidate active substances, has the advantages of low cost and high efficiency, and has important application value.

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of circ0005704 and related biological elements thereof in diagnosis and treatment of recurrent spontaneous abortion with unknown reasons. Researches find that differential expression and autophagy level of ULK1 in trophoblast cells of URSA patients are increased. High-throughput transcriptome sequencing is combined with bioinformatics analysis, and it is found that miR-26a-5p is down-regulated in URSA patients, is responsible for up-regulation of ULK1 and promotes autophagy of trophoblast cells, so that occurrence of URSA is increased. Through a high-throughput transcriptional set screening strategy and dual luciferase reporter gene analysis, it is found that circ0005704 enhances the expression of ULK1 through miR-26a-5p. In a word, the research of the invention shows that the circ0005704 / miR-26a-5p / ULK1 signal axis participates in the pathogenesis of URSA by adjusting the migration of the trophoblast cells, and a new target and scientific evidence are provided for the clinical treatment of URSA.

The invention specifically discloses a salix cheilophila in-situ genetic transformation method independent of a tissue culture system, and belongs to the field of plant genetic engineering. According to the method, semi-lignified branches of salix cheilophila are taken as receptors, vacuumizing treatment is performed by utilizing agrobacterium rhizogenes infection liquid, hairy roots are directly induced under a water culture condition after short-term dark culture, and positive identification is performed through reporter genes. According to the method, tedious sterile tissue culture operation is completely avoided, the transformation period is shortened to about 22 days, and the technical threshold and the time cost are remarkably reduced. By adopting the system, the positive rate can reach 54.33% when the pBI121 vector is used for transformation, and the efficiency is remarkably improved compared with that of an existing method. The method provides an efficient and reliable technical platform for root system gene function research, expression analysis and genetic improvement of salix cheilophila and related species thereof, and has high application potential.

The invention relates to the technical field of gene engineering, in particular to a transgenic zebrafish system pTo12-efla-tjp1a-P2A-mCherry based on a Tol2 transposition subsystem and a construction method of the transgenic zebrafish system pTo12-efla-tjp1a-P2A-mCherry. The preparation method comprises the following steps: S1, constructing a Tol2 transposon expression vector pTol2-ef1a-tjp1a-P2A-mCherry which contains an ef1a promoter, a tjp1a gene, a P2A peptide sequence and an mCherry reporter gene; s2, carrying out in-vitro transcription to prepare Tol2 transposase mRNA; s3, mixing the expression vector with Tol2 transposase mRNA, and microinjecting the mixture into the single-cell stage embryo of the zebra fish; s4, performing fluorescence screening on the F0 generation embryos surviving after injection to obtain the Founder fish with positive transgenosis; s5, after the F0-generation positive fish is bred to be sexually mature, an F1 generation is obtained through mating, transgenic positive individuals with stable inheritance are screened out after identification, and a transgenic zebrafish strain is established. According to the transgenic zebrafish system pTo12-efla-tjp1a-P2A-mCherry based on the Tol2 transposon system and the construction method of the transgenic zebrafish system pTo12-efla-tjp1a-P2A-mCherry based on the Tol2 transposon system, efficient integration of exogenous genes is achieved by utilizing the Tol2 transposon system, and the transgenic zebrafish line capable of being stably inherited to the F1 generation is successfully obtained.

The invention provides a chicken PGC reporter gene cell strain for detecting the biological activity of FGF2 as well as a construction method and application of the chicken PGC reporter gene cell strain, and belongs to the technical field of biological activity detection. The cell strain is a recombinant cell which is stably integrated with a reporter gene expression cassette responding to an FGF signal channel in a chicken PGC genome; the expression cassette comprises a serum response element (SRE), a minimum promoter and a reporter gene (such as a luciferase gene). The construction method comprises the following steps: introducing a donor vector containing the elements into chicken PGC by utilizing a PiggyBac transposon system, and screening to obtain a stably expressed cell strain. The cell strain provided by the invention retains endogenous signal channel characteristics of chicken PGC, and can specifically and highly sensitively respond to FGF2 stimulation. The method can be used for biological activity quantitative determination and thermal stability evaluation of the FGF2 protein and the mutant thereof, quality control of culture medium components and screening of an FGF signal channel regulator.

The invention discloses a rubus cochinchinensis efficient genetic transformation method based on RUBY reporter gene and agrobacterium rhizogenes mediation, which realizes that the transformation period is shortened to be within 15 days and the transformation efficiency is improved to be more than 60% through direct explant treatment under a non-tissue culture condition. The method comprises the following specific steps: 1) preparing an explant material; (2) constructing an RUBY carrier and transforming agrobacterium rhizogenes; (3) infecting the explant by utilizing agrobacterium rhizogenes carrying a target gene; (4) co-culturing after infection and carrying out RUBY visual screening; 5) positive root molecular biology verification; (6) inducing positive roots into buds; according to the method, induction of the positive callus, the positive root and the positive bud of the rubus coreanus stem segment under the non-tissue culture condition is successfully achieved, a stable and efficient genetic transformation system is constructed, the method has the remarkable advantages of being easy and convenient to operate, low in cost, wide in applicability, high in reproducibility and the like, and a key technical support is provided for molecular breeding of the rubus coreanus.

A plasmid capable of automatically inserting a membrane-localized luciferase reporter gene into a chromosome of a bacterium / recipient bacterium. After the plasmid inserts a luciferase reporter gene into a chromosome of a bacterium / recipient bacterium, the plasmid can be lost. Since the plasmid can be lost, the bacterium / recipient bacterium into whose chromosome the luciferase reporter gene is inserted does not require the addition of antibiotics during subsequent use, providing convenience and reducing interference.

Provided herein are methods of sex sorting a plurality of insects based on sex-specific gene expression, the methods comprising (a) generating an exogenous nucleic acid molecule; (b) delivering the exogenous nucleic acid molecule into an insect body of the plurality of insects, wherein the exogenous nucleic acid molecule comprises a promoter region, a gender-specific splicing module, a reporter gene, and a transcription terminator; (c) detecting gender specific gene expression of the reporter gene; and (d) sorting the insects of the plurality of insects based on the detection of the gender-specific gene expression in step (c), thereby sex-sorting the insects based on the gender-specific gene expression.

There are SARS coronavirus 2 recombinant vectors derived from a GH clade SARS coronavirus 2 Korean isolate, which express distinct reporter genes, and the production method thereof. A full-length clone of a SARS coronavirus 2 Korean isolate or a derivative thereof, according to one embodiment, can be used as a standard material for evaluating the efficacy of therapeutic agents and vaccines in cell lines and animal models while maintaining infectivity and replication capacity when restored to viruses, can be used to develop a large-scale testing method for therapeutic agent development, and can be used to develop attenuated vaccine strains. In addition, a SARS coronavirus 2 recombinant vector derived from a Korean isolate or a derivative thereof, expressing a reporter gene, can be used for high-capacity, rapid drug screening in the development of antibody therapeutic agents and antiviral agents.

The invention discloses a bacillus constitutive strong promoter and application thereof, and relates to the technical field of biology, and the nucleotide sequence of the promoter is SEQ ID NO: 2. The mRNA transcriptional level of the reporter gene is improved; the method is used for starting a target gene to efficiently express amylase in bacillus subtilis. The method is also used for starting a target gene to efficiently express alkaline protease in bacillus licheniformis. The novel constitutive promoter with enhanced transcriptional activity is developed by taking P43 as a starting template, and under the same host and culture conditions, the absolute level of the constitutive promoter for driving expression of a reporter gene or a target product is higher than that of the current P43 promoter, so that core power is provided for performance improvement of a bacillus cell factory.

The invention discloses an SRCIN1 gene therapy vector for autism treatment and application of the SRCIN1 gene therapy vector, and relates to the field of autism treatment. The vector is a lentiviral vector and comprises an overexpression sequence of an SRCIN1 gene, the overexpression sequence is pSLentiti-CMV-Srcin1-P2A-EGFP-WPRE, and the titer of the overexpression sequence is 2.99 E + 08 TU / ml. The SRCIN1 gene therapy vector is delivered to the prefrontal cortex through brain stereotactic injection and is used for up-regulating SRCIN1 expression and improving social behaviors, cognitive functions and anxiety-like behaviors of autism model mice. The SRCIN1 gene therapy vector further comprises an EGFP (enhanced green fluorescent protein) reporter gene which is used for visualizing transfection efficiency. The vector disclosed by the invention is simple to prepare and high in targeting property, and can be used for remarkably improving the social ability and exploration ability of mice, improving infantile autism behaviors such as anxiety behaviors and repeated engraving behaviors of the mice and increasing PSD95 protein expression.

This invention relates to the field of plant genetic transformation technology, and provides a method for genetic transformation of crape myrtle stem segments with buds. The method includes: infecting crape myrtle explants with Agrobacterium tumefaciens containing the RUBY reporter gene, and co-culturing them in a co-culture medium; then transferring the co-cultured explants to a differentiation medium for differentiation culture; and inoculating the obtained clustered buds into a rooting medium to obtain genetically transformed positive seedlings; wherein the explants are crape myrtle tissue culture seedlings with stem segments containing buds. The crape myrtle stem segments used in this invention have a higher regeneration efficiency than leaves. The adventitious bud induction rate of crape myrtle leaves is 1.0%-4.7%, and the stem segment regeneration and proliferation coefficient is 5-20. A large number of positive plants can be obtained in the later stage, and no browning phenomenon occurs in any of the stem segments after infection.

The invention relates to a gene expression cassette and application thereof. The gene expression cassette comprises a tyrosinase gene, a transcriptional repressor protein gene, a melanin response promoter and a reporter gene, and the reporter gene is located at the downstream of the melanin response promoter and is regulated by the promoter. The gene expression cassette with a specific structure is designed, melanin in cells can be responded, a melanin biosensor can be further constructed by utilizing the gene expression cassette, the gene expression cassette has good specificity, and high-throughput screening of strains can be realized. Furthermore, ALE evolution is carried out on the strain for producing melanin, high-throughput screening is carried out based on a designed sensor, the strain for producing melanin with high yield is obtained, through shake-flask culture and 5L fermentation tank culture, the yield of melanin reaches up to 10.84 g / L and 29.04 g / L respectively, and an efficient and feasible solution is provided for large-scale production of melanin.

The present application relates to the field of genetic engineering, and in particular to a vector for analyzing plant promoter expression specificity, a preparation method and application thereof. The T-DNA region of the vector comprises, from 5' end to 3' end, a callus-specific promoter-driven selection marker gene expression cassette and a reporter gene expression cassette with a multiple cloning site; the callus-specific promoter-driven selection marker gene expression cassette comprises a callus-specific promoter, a selection marker gene coding sequence and a terminator. The vector of the present application utilizes a rice callus-specific promoter to drive the expression of a selection marker gene, can greatly reduce the non-specific interaction with the promoter of a target gene, improve the specificity of the expression of the target gene, and effectively reduce the biological safety risk caused by the selection marker gene in a transgenic plant, and has important application value in basic theoretical research and molecular breeding.

Provided herein are compositions and methods for real-time identification and isolation of base-edited cell populations. Also provided herein are methods for producing enriched isogenic lines of genetically modified cells, including base-edited human pluripotent stem cells. In particular, provided herein are methods utilizing transient expression of reporter proteins, the detectable signal of which is altered following base editing. Using the transient reporter with a base editor permits enrichment of isogenic populations of base-edited cells.

The invention discloses an enhancer regulatory element for transcriptional regulation expression of a human LTF gene and application of the enhancer regulatory element. The sequence of the regulatory element is SEQ ID No.1. Experiments prove that the human specific regulatory element can significantly activate report gene expression (improved by 14.93 times) in a bovine mammary epithelial cell line (MACT), the limitation of dependence on species conservative elements in the prior art is broken through, and an expression system designed based on the element can realize human level LTF high-efficiency expression. And a brand new tool is provided for development of mammary gland bioreactors and functional dairy products.

The invention discloses a yeast clone with a pAbAi reporter gene as well as a construction method and application thereof, the yeast clone comprises a bait sequence Bit as shown in SEQ ID NO: 1 and an original promoter Pmin as shown in SEQ ID NO: 2, the promoter of the pAbAi reporter gene is formed by the inserted Bit and Pmin, a pAbAi / -CAAT plasmid as a no-load plasmid as shown in SEQ ID NO: 3 is constructed, the bait sequence as shown in SEQ ID NO: 1 is connected, and the pAbAi reporter gene is obtained. BstBI is used for carrying out single enzyme digestion on pAbAi-Bait / CAAT plasmids, Y1Hgold yeast competent cells are converted, SD / -Ura plates are coated, after the Y1Hgold yeast competent cells grow for 3-5 days, monoclonal colonies are selected and subjected to colony PCR detection, it is confirmed that a bait sequence is successfully integrated into the Y1Hgold yeast competent cells, and subculture is carried out, so that the Y1Hgold yeast is obtained.

This invention discloses a genetic transformation and gene editing method applicable to multiple sesquiterpene species, belonging to the field of plant genetic transformation and gene editing technology. The method uses the cotyledonary nodes of sterile seedlings of the sesquiterpene genus as explants, and introduces an optimized pScEF1α-Cas9 gene editing transformation vector using Agrobacterium infection. Regenerated plants are obtained through recovery culture, selection culture, and rooting culture. The vector contains four gene expression units: pScEF1α:Cas9, pGmU6:sgRNA, pGmUBI:DsRed2, and pCaMV35S:HygR. In vivo screening is further performed using the DsRed2 fluorescent reporter gene, and the gene-edited plants are identified by molecular detection. This method is applicable to common sesquiterpene, stem-nodled sesquiterpene, and spiny sesquiterpene, and has advantages such as strong species versatility, high transformation efficiency, short cycle, visualized screening, and high positive rate, providing efficient technical support for gene function research and genetic improvement of sesquiterpene species.

This invention provides a method for constructing a fluorescently traced mouse model and its application in the sorting of Mcpt4-positive and negative mast cells. First, the Cre gene is knocked into the Mcpt4 gene sequence of C57BL / 6 mice using CRISPR / Cas9 technology. Then, through animal hybridization and Cre-loxP recombination technology, a fluorescently traced mouse model of Mcpt4-positive mast cells is obtained. The fluorescently traced mouse model possesses a dual-fluorescent reporter gene system consisting of the red fluorescent protein tdTomato and the green fluorescent protein ZsGreen. Mcpt4-positive mast cells exhibit red fluorescence, while other Mcpt4-negative cells exhibit green fluorescence. Using this fluorescently traced mouse model, Mcpt4-positive and negative mast cells can be rapidly, accurately, and effectively sorted, providing a reliable technical basis for exploring the roles and mechanisms of Mcpt4-positive and negative mast cells in immune responses.

The invention discloses a method for screening and identifying ribosome entry sites in a short chain, which comprises the following steps of: (1) screening a short chain IRES sequence with the length of less than 100nt from an IRES database, constructing a screening library and synthesizing; (2) constructing a screening vector containing double reporter genes, cloning the short-chain IRES into a linearized screening vector, and generating circular RNA which only depends on the short-chain IRES sequence to recruit ribosome expression of the reporter genes in cells by the screening vector cloned with the short-chain IRES sequence; and (3) carrying out cell transfection on the screening vector cloned with the short-chain IRES sequence, screening out cells expressing reporter genes through cell sorting, and sequencing through a ribosome-newborn peptide chain compound to obtain the short-chain IRES sequence capable of recruiting ribosome to start circular RNA translation in the cells. According to the invention, a short-chain IRES sequence library is constructed, and short IRES capable of starting circular RNA translation is screened, so that transfection limitation caused by overlarge molecular weight of the current IRES sequence is overcome.

The present invention relates to novel cell lines for the expression of the human LHCG receptor and for the expression of the human LHCG receptor and a luciferase reporter gene. Methods for producing such cell lines are also described. The cell lines of the present invention, which express the LHCG receptor and a luciferase reporter gene, can be used in methods for determining the presence and biological activity of r-hLH and r-hCG samples.

In one aspect, a method of treating an NK cell to induce a gene edit. The method may include contacting an NK cell with a transfection composition including poly-L-glutamic acid (y- PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof; contacting the NK cell with an editing composition including: a DNA-PK inhibitor, and an HD AC inhibitor; electroporating the NK cell; and contacting the NK cell with a recovery composition including DNase I. In another aspect, a method of preparing an engineered NK cell comprising a fluorescent reporter at an endogenous locus. In another aspect, a method of treating an NK cell to introduce an exogenous nucleic acid.

The invention relates to the technical field of biomedicine, and particularly provides a mouse model capable of being inductively combined with epithelial cell specific ablation and application of the mouse model. The method comprises the following steps: inserting a '3xFLAG-P2A-CreERT2 'functional frame at a fixed point at a termination codon of a mouse Odam gene, so as to obtain an OdamCreERT2 mouse. The expression specificity of the Odam gene in binding epithelium is verified by a mouse obtained by copulating with a ROSA26mT / mG reporter gene mouse; mice obtained by mating with ROSA26DTA cell ablation mice reveal the indispensable function of binding epithelium in maintaining periodontal barrier and preventing bone resorption. The model retains endogenous Odam expression and functions, supports protein dynamic tracing and space-time specific gene manipulation, and is suitable for researching the effect of binding epithelium in periodontal barrier integrity and alveolar bone resorption prevention.

This invention relates to a pyridoxal phosphate-responsive biosensor and its application, belonging to the field of synthetic biology biosensor technology. This invention is the first to discover and identify the relationship between pyridoxal phosphate and Cgl1185-P. cgl2838 The transcriptional regulation and response relationship between these factors was investigated, and this relationship was used as the basis for constructing a biosensor responsive to pyridoxal. The transcription factor regulatory system Cgl1185-P derived from Corynebacterium glutamicum was used. cgl2838 A biosensor for the specific detection of pyridoxal phosphate was constructed by assembling a reporter gene as the main component onto a plasmid, enabling effective detection of pyridoxal phosphate concentrations in intracellular or culture environments. This biosensor possesses advantages such as clear structure, high sensitivity, and good host adaptability, and can be used for fermentation process monitoring, cofactor metabolism research, and screening of relevant strains, showing promising application prospects in the fields of cofactor concentration monitoring and metabolic regulation.

The invention belongs to the technical field of gene editing, and particularly relates to a WER10 enhancer and application thereof. The invention provides application of a WER10 nucleic acid molecule and / or a biological material for regulating and controlling the WER10 nucleic acid molecule, the nucleotide sequence of the WER10 nucleic acid molecule is as shown in SEQ ID NO: 1, and the application comprises any one or more of the following items: 1) application of the sequence as shown in SEQ ID NO: 1 as an enhancer; 2) application in enhancing the expression of a downstream reporter gene LUC induced by an immune induction signal; and 3) application in regulation and control of the broad-spectrum antibacterial property of the plants. The sequence and the characteristics of the pathogenic bacterium induced enhancer WER10 located in the intergene region are identified and verified from arabidopsis thaliana, plant immunity related cis-regulatory element resources are enriched, and a new resource is provided for constructing a pathogen induced disease-resistant expression system and developing crop disease-resistant molecular design and breeding.

This invention relates to the field of medical detection technology and discloses a fluorescent sensor for high-sensitivity detection of circRNA and its detection method based on machine learning. The sensor includes a splitting probe M, a splitting probe N, a circular template T, a probe CrRNA, and a reporter gene. The 5' end of probe M is modified with a phosphate group, and the 5' end of probe M and the 3' end of probe N each have 15 nucleotides complementary to the target. The single-stranded DNA of splitting probe M and the single-stranded DNA of splitting probe N are ligated into the single-stranded DNA of probe MN in the presence of the target. The 5' end of the circular template T is modified with a phosphate group, and probe T, from its 5' end to its 3' end, consists of the N probe complementary region, the functional region, and the M probe complementary region, respectively. The CrRNA probe, from its 5' end to its 3' end, consists of the protein-binding region and the functional complementary region, respectively. The 5' end of the reporter gene is modified with a fluorescent group, and the 3' end is modified with a quenching group, which quenches the fluorescence signal of the 5' end fluorescent group.

The invention discloses a method for detecting an AAV neutralizing antibody. The method comprises the following steps: preparing a recombinant AAV vector AAV-Gluc carrying a Gaussian luciferase reporter gene; the method comprises the following steps: diluting AAV-Gluc according to a certain dilution step to prepare an AAV-Gluc working solution; diluting the sample to be detected; carrying out cell planking, culturing the target cells to form a uniform cell suspension, and adding the cell suspension into a cell culture plate; mixing the sample diluent with the AAV-Gluc diluent, and carrying out incubation; adding the incubated sample mixed solution into the cell culture plate, and carrying out virus infection and culture; obtaining a cultured cell supernatant, adding a detection solution, and detecting signal intensity; and analyzing and sorting the data to obtain the neutralization titer. The secreting type Gaussian luciferase reporter vector is used, target protein is secreted outside cells after being expressed, and detection is facilitated; the infected supernatant is directly sucked for detection, and the operation is convenient and fast; cell lysis and extraction are not needed, protein loss is reduced, and detection accuracy and precision are improved.

The invention provides a nucleic acid construct which comprises an immune response regulatory sequence and a coding sequence of a reporter gene driven by a promoter sequence, and the immune response regulatory sequence comprises a transcription factor binding regulatory element RE which is co-stimulated and regulated by transcription factors AP-1 and CD28. The invention also provides an in-vitro immune effector function reporter gene cell model containing the nucleic acid construct, and the cell model can realize effector function activity determination performance of sensitive and potent signals so as to evaluate ADCC and ADCP killing action mechanisms and titers mediated by antibody-dependent disease cells designed by an Fc structural domain of a therapeutic antibody product. In addition, the immunosuppression and regulation efficacy of a targeted CTLA-4 or Abatacept mediated treatment method on a CD28 co-activation pathway can be evaluated.