8 results about "Egfp gene" patented technology

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 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 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.

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.