19 results about "MCherry" patented technology

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 application provides a construction of a fluorescent protein labeled microtubulin and a microtubule binding protein universal bivalent carrier, and the process is as follows: a first stage is to construct a GFP-alpha tubulin carrier, and a second stage is to construct a GFP-alpha tubulin-mCherry universal bivalent co-expression carrier; in the second stage, 35S, mCherry and NOS sequences are inserted into a KpnI enzyme cutting site of a multiple cloning site of the GFP-alpha tubulin carrier constructed in the first stage, and a single nucleic acid enzyme cutting site is inserted into the 5' end and the 3' end of the mCherry sequence, respectively, and the single nucleic acid enzyme cutting site is XbaI, KpnI / Acc65I and AscI sequences, respectively, so as to obtain the universal bivalent carrier. The universal bivalent carrier provided by the application can express two target genes simultaneously, has the fluorescent signals of GFP and mCherry, can quickly and accurately identify a transgenic plant, is convenient for positive seedling screening, can be used for a tobacco transient expression experiment, can shorten an experimental period, and saves time and effort.

The invention discloses a genetically engineered bacterium for producing D-pantothenic acid based on a corynebacterium glutamicum CPSPC system as well as a construction method and application of the genetically engineered bacterium. Wild type C.glutamicum ATCC 13032 is taken as a chassis strain, and a functional combination of a glycine ribosome switch BsGR from bacillus subtilis and a promoter Ptuf / Ph36 is verified through fluorescent protein mCherry; a GCS system of escherichia coli and bacillus subtilis is introduced to the strain DPAg-15, genes related to synthesis of 5, 10-dimethyltetrahydrofolic acid are tested, it is found that serA delta197GTG / ATG significantly increases the yield of D-pantothenic acid, the yield of D-pantothenic acid of the constructed strain DPAj-2-tuf is increased to 23.06 g / L (63 h), the yield is 0.37 g / L / h, and the conversion rate is 0.17 g / g; the engineering strain DPAj-2-tuf is obtained by dynamically regulating and controlling a D-pantothenic acid synthesis path through a CPSPC system and combining plasmid overexpression serA delta197GTG / ATG, the yield of the engineering strain DPAj-2-tuf is increased by 2.1 times compared with that of a traditional method, and an efficient technical scheme is provided for producing D-pantothenic acid through a microbial fermentation method.

The invention relates to the technical field of saccharomyces cerevisiae expression foreign protein, and discloses a saccharomyces cerevisiae strain with ASFV p14.5 displayed on the surface and a construction method and application thereof, and the method comprises the following steps: (1) synthesizing a delta site homologous arm sequence; (2) seamlessly connecting the homologous arm sequence of the delta site to a carrier pET23a, connecting a selective marker tryptophan biosynthetic gene TRP1 to the 5'end of the upstream homologous arm sequence of the delta site, and sequentially connecting an Aga2 gene, an E120R gene, a T2A peptide gene and an mCherry gene to the 5 'end; (3) carrying out enzyme digestion on two sides of the delta site through restriction endonuclease salI, and recovering a linearized fragment through agarose gel; and (4) integrating the recovered fragments into a host saccharomyces cerevisiae genome through a chemical conversion method, and culturing until bacterial colonies grow. The surface display type saccharomyces cerevisiae strain obtained by the invention not only has the stability of integrative saccharomyces cerevisiae for expressing foreign protein, but also can be used for more easily and more intuitively screening a high-expression quantity saccharomyces cerevisiae strain by combining the T2A peptide with mCherry.

This invention belongs to the field of genetic engineering technology, specifically disclosing a method for preparing and applying gene-modified homozygous cells of chicken progenitor cells (PGCs). Using a dual-fluorescent marker gene, two donor plasmids carrying green fluorescent protein (EGFP) and red fluorescent protein (mCherry), respectively, are transfected into PGCs. Cells simultaneously expressing red and green fluorescence are obtained using flow cytometry, thus forming homozygous cell lines. Then, the dual-fluorescent marker gene is deleted using the Cre-LoxP system combined with electroporation, and residual LoxP fragments are deleted using ssODN as a donor template, achieving traceless gene editing in recipient chicken embryos. This invention does not introduce any exogenous genes, increasing the safety of gene-modified chickens and is of great significance for eliminating the biosafety risks associated with gene-edited chickens.

The application belongs to the technical field of biology, and particularly relates to a mitochondrion function evaluation method and application thereof. The application provides an innovative mitochondrion function evaluation method, which reports the ATP concentration change in the mitochondrion matrix and the mitochondrion morphology in real time, in situ and quantitatively through ATP response fluorescent protein Mito-AT1.03 with a mitochondrion positioning signal, directly reflects the mitochondrion energy metabolism and the mitochondrion fusion / fission state; meanwhile, a fusion protein Parkin-mCherry is constructed by using Parkin protein, a key starting factor of mitochondrion autophagy, as an early specific mark of autophagy initiation. The two are co-transfected into cells, and the signal level and positioning condition of the two fluorescent proteins are observed, so that the mitochondrion function can be accurately and timely evaluated, and a new idea is provided for the mechanism research of mitochondrion dysfunction and the methodological development of the screening of mitochondrion function regulators.

A method for constructing a mouse model for rapid degradation of Myostatin (MSTN) protein is provided. The method includes: inserting sequentially a 5′ homologous arm, 3 X (Glu-Ala-Ala-Ala-Lys) (3xEAAAK), an FK506-binding protein (FKBP1A) gene synonymous mutation sequence, 3xEAAAK, a red fluorescent protein monomeric Cherry (mCherry) sequence and a 3′ homologous arm into a Donor vector backbone to obtain a Donor recombinant vector; designing a Guide Ribonucleic Acid (gRNA) based on a third exon of the Mstn gene; mixing the gRNA, CRISPR-associated protein 9 (Cas9) protein, and the Donor recombinant vector and co-injecting a mixture of the above into mouse fertilized eggs, culturing the fertilized eggs in vitro and transplanting to obtain the F0 generation mice; and hybridizing the F0 generation mice with wild-type mice, or further breeding hybridized offspring to obtain a knock-in mouse model.

The application discloses a method for constructing a fluorescently labeled piwil1 gene knockout sterile zebrafish line, which utilizes a CRISPR / Cas9 system to construct a piwil1 gene knock-in vector carrying P2A-EGFP or P2A-mCherry, respectively, to obtain two hybrid strains through microinjection, and then to screen a homozygote expressing EGFP and mCherry simultaneously by mating the two strains. The homozygote shows male sterility and can be used for reproductive development research and ecological prevention and control of transgenic fish. The application realizes visual labeling of the function of the piwil1 gene and accurate screening of the sterile phenotype, and is simple and efficient to operate.

The invention relates to the technical field of genetic engineering and model animals, in particular to a visual myocardial specific gene knockout zebrafish model and a construction method and application thereof. According to the technical scheme, the visual myocardial specific gene knockout zebrafish model comprises a model body, the model body is transgenic zebrafish, and a genome of the model body comprises a Cas9 protein coding sequence driven by a myocardial specific promoter; a Tg transgenic zebrafish strain is constructed, a cmlc2 promoter with high cardiac muscle cell specificity is utilized, expression of Cas9 protein optimized by zebrafish codons and expression of red fluorescent protein mCherry are driven at the same time, the Cas9 protein and the red fluorescent protein mCherry are connected through a P2A self-cleavage peptide sequence, it is ensured that independent and complete functional protein is generated through translation after transcription, and the expression of the Cas9 protein and the red fluorescent protein mCherry is promoted. The expression of the Cas9 protein is completely coupled with an mCherry fluorescence signal.

The present application relates to a kind of construction method of macrophage specific fluorescent reporter mouse model, belong to genetic engineering and genetic modification technical field.The present application is first by CRISPR / Cas9 genome editing technology to C1qc gene site is edited after obtaining a kind of brand-new C1qc-CreChy gene knock-in mouse model, realizes the expression of Cre and mCherry is directly driven by C1qc promoter, and it is verified by flow cytometry that mCherry only in the microglia of mouse brain and the macrophage in spleen has very high expression, and mCherry expression is not detected in other immune cells, to realize the tracing of the two kinds of macrophages, for studying its function.The present application provides a kind of usable animal model for studying the heterogeneity of macrophage, and provides a kind of new animal model for studying the complex immune function in body.

The invention relates to the field of vaccine preparation, and discloses a saccharomyces cerevisiae strain for displaying African swine fever virus protein on the surface and a construction method and application of the saccharomyces cerevisiae strain. Sequentially connecting TRP1, Aga2, African swine fever virus protein, T2A peptide and gene segments of mCherry to the 3'end of the upstream homologous arm sequence of the delta site to obtain a recombinant plasmid; and (2) carrying out enzyme digestion on upstream and downstream regions of the delta site by using incision enzyme, separating by adopting agarose gel, transforming the obtained target DNA fragment onto a saccharomyces cerevisiae genome, culturing to obtain a transformed bacterial colony, and observing a single bacterial colony with red fluorescence, namely the saccharomyces cerevisiae strain with the surface displaying the African swine fever virus protein. The saccharomyces cerevisiae strain capable of displaying ASFV p54 or ASFV j18L on the surface is successfully constructed, the strain not only can stably express ASFV protein, but also can rapidly screen positive clone through red fluorescence, and the protein expression verification mode is simple.

The invention provides a high-fidelity Cas9 variant, a tissue targeting lipid nanoparticle delivery system, a CRISPR-Cas9-mediated genome precise editing method and application thereof, and belongs to the technical field of genetic engineering, the high-fidelity Cas9 variant HyperFi-Cas9 is adopted, four mutations of R691A, N497A, R661A and Q695A are introduced, and the off-target effect is reduced to 0.025% or below while the wild type targeting activity is kept not lower than 85%; a tissue targeting intelligent lipid nanoparticle delivery system is adopted, and by optimizing lipid composition and ligand modification, 30-60% of cell delivery efficiency and 16-80% of in-vivo editing efficiency are achieved; the integrated double-fluorescence real-time monitoring system comprises an FRET (fluorescence resonance energy transfer) molecular beacon probe and an mCherry-P2A-EGFP (enhanced green fluorescent protein) integrated report module, and is used for realizing dynamic monitoring and quantitative evaluation of an editing process; and a homologous recombination enhancement strategy is adopted.

The present application relates to the field of bioengineering, and particularly relates to a kind of artificial codon optimization nucleic acid molecule of the red fluorescent protein coding.The present application provides nucleic acid molecule of red fluorescent protein coding, and the red fluorescent protein is mCherry, compared with the existing red fluorescent protein nucleotide sequence, the number of CpG site is increased from 56 to 92, the content of the third position of codon G / C (GC3) is not less than 99%.The nucleotide sequence provided by the present application is the mCherry synonymous codon substitution sequence obtained by combining the increase of the number of CpG sites and the use of optimal codon strategy, which is 1.3-2.4 times higher than the fluorescence intensity of the sequence before modification in mammalian (human / mouse) cells;The present application uses the method of codon optimization to obtain the nucleic acid molecule of the red fluorescent protein that does not exist in nature, which can encode the red fluorescent protein with stronger fluorescence intensity, and promotes the development of live cell imaging.

The invention relates to a high-performance RNA (Ribonucleic Acid) off-target detection report system as well as a construction method and application thereof. According to the invention, several modules of an MS2-MCP system, deoxyadenosine deaminase, mCherry and BFP are fused, and a fluorescence report system for indirectly detecting the miss of deoxyadenosine deaminase RNA through fluorescence and a sequencing report system for directly detecting the miss of deoxyadenosine deaminase RNA through sequencing are developed. The RNA off-target detection report system comprises an RNA off-target fluorescence report system and an RNA off-target sequencing report system. The RNA off-target detection report system can be effectively applied to RNA off-target risk assessment of the ABE base editing tool. The RNA off-target detection report system obtained through the method is convenient to operate, short in period, low in cost and high in sensitivity, effectively makes up for the shortages of RNA off-target detection in the current base editing field, and has very wide application prospects.

The invention relates to the technical field of gene editing, and discloses a gene editing efficiency detection vector based on double-color fluorescent protein as well as a construction method and application thereof. The construction method comprises the following steps: synthesizing a full-length DNA fragment containing a specific targeting sequence, an mCherry reporter gene and a C-terminal fused PEST degrading peptide sequence; performing double enzyme digestion and purification to obtain a target fragment; connecting the target fragment with the vector skeleton subjected to enzyme digestion linearization through T4 DNA ligase; the gene editing efficiency detection vector pLV3-CMV-RGFP-PEST-Neo is prepared by the following steps: carrying out gene editing efficiency detection on the gene editing efficiency vector pLV3-CMV-RGFP-PEST-Neo, transforming the gene editing efficiency vector pLV3-CMV-RGFP- The system containing the gene editing efficiency detection vector co-expresses red and green, during editing, an mCherry coding sequence is cut off, cells are converted from double positive (yellow) to single positive (green), cell lysis is not needed, the editing efficiency is quantified by calculating the change of the green / red fluorescence ratio, and the method is convenient and fast.

The application belongs to the technical field of forest tree genetic engineering, and discloses a genetic transformation method of Hippophae rhamnoides L. mediated by Agrobacterium LBA4404. The method comprises the following steps: flower branch selection, activation and culture of Agrobacterium, preparation of Agrobacterium transformation solution, infection of Hippophae rhamnoides L. plants, mCherry fluorescence detection, and mCherry expression quantity detection. After the Agrobacterium LBA4404 containing the mCherry reporter gene is used to dip the female flower inflorescence of Hippophae rhamnoides L. after pollination, transgenic fruits are obtained. The method can greatly shorten the transformation period, the experimental results are relatively intuitive through fluorescence observation, and the method can be used for long-term stable expression, thereby providing a quick and reliable method for gene function verification of Hippophae rhamnoides L. and laying a foundation for cultivation of new transgenic Hippophae rhamnoides L. varieties.

This invention discloses a method for producing and identifying haploid rice, belonging to the field of biotechnology. The method includes the following steps: (1) using a dominant male-sterile line as the female parent and a haploid inducible line carrying the red fluorescent protein gene mCherry as the male parent for hybridization and pollination to obtain hybrid seeds; (2) after the hybrid seeds germinate, candidate haploid seeds without fluorescence are screened by observing the red fluorescence in the roots and buds; (3) the seedlings obtained from the candidate haploid seeds are identified by flow cytometry. This invention uses a dominant male-sterile line as the hybrid female parent, which improves the efficiency of pollination and hybridization and greatly reduces the amount of manual labor and time; the haploid inducible line combined with fluorescent labeling technology, by screening for non-fluorescent seeds and then performing flow cytometry background measurement to determine ploidy, haploid seedlings can be screened in the early stage, and the screening efficiency is greatly improved, while saving time and field planting workload.

The invention relates to the technical field of biology, in particular to an insulating double-fluorescence reporting system for quantitatively measuring terminator efficiency and a construction method and application thereof. According to the method, RiboJ ribozyme is introduced, mRNA is automatically cut after transcription to eliminate errors caused by translation initial efficiency differences, a stable endogenous promoter is adopted to control fluorescent protein mCherry expression, internal initiation codon removal optimization is carried out on fluorescent protein, generation of truncated protein is avoided, and signal reliability is improved. The method is suitable for various prokaryotic hosts, the terminator efficiency from weak to strong can be accurately measured, the dynamic range is wide, the sensitivity is high, only conventional molecular cloning and flow cytometry are needed in operation, and an efficient and accurate evaluation method is provided for the terminator efficiency.

The application discloses a recombinant protein for detecting paraneoplastic Yo antibody by CBA method and application, and belongs to the technical field of biological medicine and engineering. The amino acid sequence of the recombinant protein comprises, in sequence, a secretion signal peptide, a CDR2 protein partial sequence, a CDR2L protein partial sequence, a transmembrane region and a fluorescent label. The secretion signal peptide is a human albumin signal peptide ALB. The transmembrane region is CD8a hinge. The fluorescent label is mCherry. The application constructs a novel recombinant protein which can be stably overexpressed on the cell membrane of eukaryotic cells by intercepting specific partial sequences of CDR2 protein and CDR2L protein, and redesigning and fusing a fluorescent label by using a secretion signal peptide, a transmembrane sequence and a connecting peptide. The recombinant protein retains the core antigen regions of the two, can effectively overcome the insufficiency in sensitivity and specificity, can significantly improve the detection rate of paraneoplastic Yo antibody when CBA method is used for detection, and can reduce the false positive rate, so that the requirement of clinical detection can be better met.