18 results about "Egfp gene" patented technology

The invention provides application of a porcine parainfluenza virus type 5 as an expression vector, and belongs to the technical field of biological vaccines. According to the invention, a PIV5 strain separated from diarrhea pigs in Jiangsu Province in China is utilized to construct PIV5 virus infectious clone. The eGFP gene is inserted into a PIV5 infectious cloned virus genome, the recombinant PIV5 virus capable of expressing the green fluorescent protein is successfully rescued, and when the eGFP gene is inserted into a P / M gene spacer region, the expression quantity is the highest, which indicates that an exogenous gene expression cassette can influence the expression level of an exogenous gene, and the P / M gene spacer region is the optimal site for inserting the exogenous gene, so that the recombinant PIV5 virus can be successfully rescued. And a solid foundation is laid for developing a vector vaccine by utilizing the swine PIV5. The porcine parainfluenza virus type 5 can be used as an expression vector to express foreign proteins, can also be used for preparing oral vaccines, and can relieve the stress reaction of pigs in the immune process.

The present invention discloses an Ush1c gene enhancer and its application, belonging to the field of genetic engineering, and the nucleotide sequence of the enhancer is shown in SEQ ID No. 1. The mouse embryo microinjection system is used to confirm that the sequence is an enhancer, which can significantly enhance the transcription activity of the EGFP protein gene in the inner ear. Compared with other sequences of similar size, the enhancer sequence fragment can drive the reporter gene or Ush1c to express significantly in the inner ear, and specifically express it in inner and outer hair cells, and is suitable for recombinant DNA, vectors or adenovirus and other biological materials, and is used to promote the transcription of EGFP gene or other genes in the inner ear.

The invention discloses a chicken sex identification method based on fluorescence labeling, and belongs to the technical field of gene engineering. The chicken sex identification method comprises the following steps: co-transfecting a CRISPR / Cas9 system and a WPG-EGFP donor plasmid into chicken primordial germ cells PGCs, screening to obtain EGFP positive PGCs, micro-injecting the EGFP positive PGCs into a receptor chicken embryo, hatching to obtain an EGFP reproductive chimera chicken, and identifying the sex through fluorescence labeling, the CRISPR / Cas9 system comprises sgRNA of a targeted chicken W chromosome WPG gene; the WPG-EGFP donor plasmid is constructed by inserting an EGFP gene into a WPG gene locus through a homologous recombination strategy. According to the method, the sex of the chicken can be visually and rapidly identified. The method solves the problems that a traditional sex identification method is complex in operation and depends on sample ages, has the advantages of high efficiency and accuracy, and is suitable for sex control and screening of poultry such as laying hens and broilers.

The present invention discloses a Mitf gene enhancer and its application in improving the gene-specific expression of cochlear stria vascular cells, and relates to the field of gene expression and regulation technology. The nucleotide sequence of the enhancer is shown in SEQ ID No. 1. The mouse embryo microinjection system was used to confirm that the sequence is an enhancer, which can significantly enhance the transcription activity of the EGFP protein gene in the cochlear stria vascular cells. Compared with other sequences of similar size, the enhancer sequence fragment can drive the reporter gene or Mitf to be specifically expressed in the cochlear stria vascular cells and the expression level is significantly enhanced. The enhancer is suitable for biological materials such as recombinant DNA, vectors or adenoviruses, and is used to promote the transcription of the EGFP gene or other genes in the cochlear stria vascular cells.

The invention discloses an in-vitro transcription system of theileria annulata transformed cells and a construction method of the in-vitro transcription system, the in-vitro transcription system comprises IVT-EGFP-mRNA, and the structure of the IVT-EGFP-mRNA is 5 'UTR-Cl-Kozak-EGFP-Pacl-3' UTR-polyA (120). According to the invention, mRNA for coding the EGFP gene is synthesized through in-vitro transcription, through capping, purification and agarose gel electrophoresis analysis, the expression condition of the target gene in TaNM1 cells is finally verified through an in-vitro transfection test and an indirect immunofluorescence technology, and the result shows that the in-vitro transcription system transfected and coded with the EGFP successfully expresses the EGFP, and the expression condition of the target gene in the TaNM1 cells is verified. The invention shows that a system for transfecting the EGFP gene into the theileria annulata transformed cell TaNM1 through an in-vitro transcription method is successfully established, and an important reference value is provided for design and research and development of theileria annulata RNA vaccines.

The invention discloses a Pcdh15 gene enhancer sequence and application thereof, and belongs to the field of gene engineering, and the nucleotide sequence of the enhancer is shown as SEQ ID No.1. A mouse embryo microinjection system is used for verifying that the sequence is an enhancer, and the transcriptional activity of EGFP protein genes in hair cells can be remarkably enhanced in inner ears. Compared with other sequences with similar sizes, the enhancer sequence fragment can drive the expression ability of a reporter gene or a Pcdh15 gene to be obviously enhanced in inner ear hair cells, is specifically expressed in inner and outer hair cells, is suitable for biological materials such as recombinant DNA (Deoxyribonucleic Acid), vectors or adenoviruses and the like, and has a wide application prospect. The method is used for promoting transcription of EGFP genes or other genes in internal and external hair cells of inner ears.

The application provides sgRNA which specifically targets the Rosa26 safe site of a cashmere goat and application of using CRISPR / Cas9 to complete site-directed knockout and site-directed integration of an EGFP gene. The application firstly uses a bioinformatics method to predict the complete sequence of the Rosa26 site, then designs two sgRNA aiming at the site, constructs a targeting vector based on the CRISPR / Cas9 system, verifies the guiding efficiency of the sgRNA, simultaneously constructs a homologous integration EGFP gene vector, co-transfects the sgRNA and the homologous integration vector into cashmere goat fetal fibroblasts, and obtains a cell strain of the Rosa26 site site-directed integration of the EGFP gene. The efficiency of the sgRNA of the application in specifically guiding Cas9 to cut the Rosa26 site reaches about 40%, effectively reduces the off-target phenomenon existing in the CRISPR / Cas9 system, and further reduces the mutation of non-target gene sequences caused by non-specific cutting. The Cas9 / gRNA expression vector can realize the specific knockout or knock-in of the Rosa26 site at the cell, embryo or even individual level, so as to study the expression of specific genes and provide technical support for the cultivation of new goat breeds.

The present invention provides an sgRNA specifically targeting a cashmere goat safety site H11 and an application of site-directed knockout and site-directed integration of an EGFP gene mediated by a CRISPR / Cas9 system. The present invention first predicts the complete sequence of the H11 site (SEQIDNO.1) using a bioinformatics method, and then designs two sgRNAs (SEQIDNO.2 and 3) for the H11 site, and the DNA sequence of the sgRNA sequence transcribed is shown in SEQIDNO.4 or 5. A targeting vector based on the CRISPR / Cas9 system is constructed, and then the guiding efficiency of the sgRNA is verified by TA cloning sequencing, and a homologous integration EGFP gene vector is constructed at the same time, and finally the screened sgRNA (Cas9 / gRNA co-expression vector) and the homologous integration vector (H11-EGFP) are simultaneously transfected into cashmere goat fetal fibroblasts to obtain a cell line of the H11 site site-directed integration of the EGFP gene. The sgRNA of the present invention can specifically recognize the H11 site of cashmere goats, guide the Cas9 endonuclease to efficiently cut the site, and the targeting efficiency is above 50%, which can effectively reduce the off-target phenomenon in the CRISPR / Cas9 system, thereby reducing the mutation of non-target gene sequences caused by non-specific cutting. By constructing a Cas9 / gRNA expression vector, the specific modification (knockout or knock-in) of the H11 site can be achieved at the cell, embryo, or even individual level, thereby studying the expression of specific genes and providing technical support for the breeding of new breeds of goats.

The application provides a construction and application of a recombinant pseudorabies virus strain stably and efficiently expressing green fluorescent protein, in the research, a green fluorescent protein gene (EGFP) is inserted into different positions of a double gene deletion vaccine strain (JS-2012-△gE / gI) of a pseudorabies virus mutant strain by using a homologous recombination method. Finally, it is proved that the recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP has good genetic stability, and the EGFP gene can also be stably and efficiently expressed, and the growth rate on cells is also comparable to that of the parent virus, so that the VP1 / 2 gene is the best exogenous gene insertion site. Subsequently, the classical swine fever virus E2 gene, the parvovirus VP2 gene, the cap gene of the circovirus type 2 and the VP1 gene of the foot-and-mouth disease virus can be replaced or inserted into the EGFP expression frame in a single or combined manner to construct different types of multi-vaccines, and the multi-vaccines have extremely high research value and platform significance.

The application discloses an sgRNA capable of effectively editing a pig CCR5 gene and application thereof, and is premised on an sgRNA capable of specifically recognizing a pig CCR5 gene in a pig genome, and successfully constructs a pig fetal fibroblast cell, a pig kidney cell line PK15 cell line and a pig 3D4 / 21 cell line with EGFP site-directed integration by using CRISPR / Cas9 mediated gene knock-in technology, and the results show that the EGFP gene can be stably and efficiently expressed in the three kinds of cells or cell lines. The application provides more site selection and reference for gene function research, gene modified pig research and development.

The invention relates to the technical field of gene engineering, in particular to a Cilp gene enhancer and application thereof. Through integration and verification of an ATAC-seq map, a single cell transcriptome data set and multi-omics data, the Cilp gene enhancer is obtained through screening, and a mouse inner ear microinjection system is utilized to further prove that the Cilp gene enhancer can significantly enhance the transcriptional activity of an EGFP protein gene in cochlea type 2 neuronal cells. Compared with other sequences with similar sizes, the Cilp gene enhancer provided by the invention can drive specific expression of a reporter gene or a Cilp gene in cochlear neurons, especially type 2 neurons, and the gene expression level is remarkably improved. The Cilp gene enhancer provided by the invention is suitable for biological materials such as recombinant DNA (deoxyribonucleic acid), vectors or adenoviruses, can be used for promoting transcription of EGFP (enhanced green fluorescent protein) genes or other genes in cochlea neurons, and provides a new targeting tool for gene therapy of hereditary hearing loss.

The invention discloses a construction method of an IER2-T2A-EGFP gene knock-in mouse model and application of the IER2-T2A-EGFP gene knock-in mouse model in myeloid cell sorting, and the method comprises the following steps: aiming at a knock-in target of a mouse IER2 gene, designing sgRNA with a sequence as shown in SEQ ID No: 2; the constructed PX459-sgRNA recombinant plasmid is used as a template, and a single-chain sgRNA + tracr RNA is obtained through PCR (Polymerase Chain Reaction) amplification and in-vitro transcription; synthesizing a Donor sequence containing the T2A-EGFP gene in an overlapping PCR (Polymerase Chain Reaction) mode; a single-chain sgRNA + tracr RNA, a Donor sequence and Cas9 protein are co-injected into male pronucleus of a mouse fertilized egg, and a female mouse is transplanted for inoculation, so that the F0-generation IER2-T2A-EGFP gene knock-in mouse model is obtained. According to the method, the primary myeloid cells can be quickly, conveniently and accurately sorted.

The invention discloses a method for fixed-point integration of a fluorescence report at a duck W chromosome EE0.6 site, and relates to the technical field of gene editing. The method comprises the following steps: transfecting an exogenous report element, an sgRNA expression vector and an spCas9-Puro expression vector into duck cells; the sgRNA expression vector comprises sgRNA of an EE0.6 site in a duck W chromosome in a targeting manner. The exogenous reporter element comprises a sequence A, an EF1 alpha promoter, an eGFP (enhanced green fluorescent protein) gene, an SV40 polyadenylic acid polyA, an SV40 promoter, a Neo resistance gene, an HSV-TK polyadenylic acid polyA, the sequence A and an sgRNA (single guide ribonucleic acid) expression element which is started by an hU6 promoter and aims at the sequence A, which are connected in sequence. According to the method, the exogenous eGFP gene can be efficiently integrated to the duck W chromosome EE0.6 site in a site-specific manner through an NHEJ way, and the method has important significance in duck sex identification and deep scientific research.

The invention provides a reporter plasmid system for multi-gene fixed-point knock-in cells and a screening method. Specifically, the invention provides a set of multi-gene knock-in reporter plasmid system which comprises sgRNA plasmids of a plurality of target genes, a plurality of genome donor plasmids, a target gene knock-in reporter plasmid, a reporter plasmid donor plasmid and a Cas9 plasmid. Wherein the reporter plasmid comprises a truncated eGFP (enhanced green fluorescent protein) gene, a target site sequence is introduced, and a gene knock-in event is accurately reported. The invention further discloses a screening method which is a method for screening simultaneous knock-in of multiple genes only through one target gene knock-in reporter plasmid during multi-gene knock-in editing, and the efficiency of sorting gene homozygous knock-in cells is improved. The method can be efficiently used for gene knock-in through HDR repair, including accurate point mutation and large fragment insertion or deletion, and a clean gene knock-in cell line without residues in a report system is obtained.

The present invention provides a recombinant baculovirus, a construction method thereof and an application. The recombinant baculovirus is obtained by integrating the gp64<supgt;TM< / supgt> <supgt;double< / supgt>-egfp gene into the genome of the baculovirus - Autographa californica nucleopolyhedrovirus (AcMNPV) through transposon recombination. The present invention also provides a construction method of the recombinant baculovirus, including obtaining the gp64 gene from the AcMNPV genome, doubling the sequence encoding the transmembrane domain (TM) in the gp64 gene to obtain gp64<supgt;TM< / supgt> <supgt;double< / supgt>, recombining gp64<supgt;TM< / supgt> <supgt;double< / supgt> into pFastBacDual-egfp, and obtaining the recombinant baculovirus AcMNPV-gp64<supgt;TM< / supgt> <supgt;double< / supgt>-egfp through the Bac-to-Bac system. Compared with AcMNPV-egfp, the AcMNPV-gp64<supgt;TM< / supgt> <supgt;double< / supgt>-egfp virus has a significantly improved proliferation ability in host cells and can reduce the median lethal time for Spodoptera frugiperda larvae. The recombinant baculovirus of the present invention has an application prospect in the preparation of microbial insecticides for controlling Spodoptera frugiperda larvae.

The invention provides construction and application of a recombinant pseudorabies virus strain capable of stably and efficiently expressing green fluorescent protein, which is characterized in that green fluorescent protein (EGFP) genes are respectively inserted into different positions of a pseudorabies virus variant double-gene deletion vaccine strain (JS-2012-gE / gI) by using a homologous recombination method. Finally, the recombinant virus JS-2012-gE / gI-VP1 / 2-EGFP has good genetic stability, the EGFP gene can be stably and efficiently expressed, and the proliferation rate of the EGFP gene on a cell is equivalent to that of a parent virus, so that the VP1 / 2 gene is an optimal exogenous gene insertion site. The classical swine fever virus E2 gene, the parvovirus VP2 gene, the cap gene of the circovirus type 2 and the VP1 gene of the foot-and-mouth disease virus can be subsequently replaced or inserted into an EGFP expression cassette in an independent or combined manner to construct different types of multi-combined vaccines, so that the multi-combined vaccine has extremely high research value and platform significance.

This invention discloses a lineage tracing method for early sheep embryonic development based on the CRISPR-Cas9 system and the PB transposon system. Specifically, it involves CRISPR-Cas9-mediated knockout of the sheep Rosa26 gene and site-specific insertion of the Cas9-T2A-EGFP gene to edit synthetic target DNA. This invention also provides a lineage tracing vector mediated by the synthetic target DNA. This vector contains 40 integrated barcodes, the synthetic target DNA, and three corresponding independent transcribed sgRNAs. The synthetic target DNA is embedded in a PB transposon vector expressing the red fluorescent protein tdTomato. This system can generate a large number of heritable repair results, and these sequences can be distinguished by the integrated barcodes. This lineage tracing vector system can be directly transfected into target cells, and the target cells can be tracked as development progresses, providing a comprehensive molecular-cellular atlas of ruminant development, with broad application prospects.

The application discloses an in vitro transcription system of a transformed cell of a circular theileria and a construction method thereof, and the in vitro transcription system comprises IVT-EGFP-mRNA, and the structure is 5'UTR-Cla1-Kozak-EGFP-PacI-3'UTR-polyA(120). The mRNA coding the EGFP gene is synthesized by in vitro transcription, and is subjected to capping, purification and agarose gel electrophoresis analysis. Finally, the expression of the target gene in the TaNM1 cell is verified by in vitro transfection test and indirect immunofluorescence technology. The result shows that the green fluorescent protein EGFP is successfully expressed by the in vitro transcription system coding the EGFP, which indicates that the system for transfecting the EGFP gene into the transformed cell TaNM1 of the circular theileria by the in vitro transcription method is successfully established, and the system provides important reference value for the design and research and development of the RNA vaccine of theileria annulata.