138 results about "Gene replacement" patented technology

An inactive CRISPR/Cas system-based fusion protein and its applications in gene editing are disclosed. More particularly, chimeric fusion proteins including an inCas fused to a DNA modifying enzyme and methods of using the chimeric fusion proteins in gene editing are disclosed. The methods can be used to induce double-strand breaks and single-strand nicks in target DNAs, to generate gene disruptions, deletions, point mutations, gene replacements, insertions, inversions and other modifications of a genomic DNA within cells and organisms.

Disclosed herein are methods for generating recombinant DNA molecules in cells using homologous recombination mediated by recombinases and similar proteins. The methods promote high efficiency homologous recombination in bacterial cells, and in eukaryotic cells such as mammalian cells. The methods are useful for cloning, the generation of transgenic and knockout animals, and gene replacement. The methods are also useful for subcloning large DNA fragments without the need for restriction enzymes. The methods are also useful for repairing single or multiple base mutations to wild type or creating specific mutations in the genome. Also disclosed are bacterial strains and vectors which are useful for high-efficiency homologous recombination.

Highly efficient gene integration or gene replacement in the higher eucaryote including animal cells can be performed by using mutant loxP site having the following properties (a)-(c) in the present invention.(a) a nucleotide sequence wherein, in a wild-type loxP site of the following formula (SEQ ID NO: 1) derived from E. coli P1 phage, at least one of the bases consisting of second (T), third (G), fourth (T) and fifth (A) bases, and at least one of the bases consisting of sixth (T) and seventh (G) bases within the 8 bases in the central part of the sequence (spacer region) are substituted by different base, and regions except for the spacer region are optionally substituted by any base:(b) a specific recombination between said mutant loxP and the wild-type loxP site can not occur even in the presence of recombinase Cre; and(c) a specific recombination between the mutant loxP sites having identical nucleotide sequences can occur in the presence of recombinase Cre.

The invention discloses a TCR (T cell receptor) gene replacement system and method mediated by retrovirus gene transfer combining with the Dual-RMCE (dual-recombinase mediated cassette exchange) technology. The TCR gene replacement system comprises a retrovirus integration vector pMP71-LGFPF containing a replacement component Loxp-EGFP-FRT, and a replacement vector pDRAV-3-LTCRF containing TCR target genes. The TCR gene replacement system has the advantages that TCR gene replacement can be performed fast in a single site of a genome, a large amount of tumor antigen specificity T cells can be produced by using the in-vivo and in-vitro cell differentiation technology, and one tumor antigen specificity TCR molecule can be expressed; the problems that retrovirus random integration causes side effects and exogenous TCRs and endogenous TCRs combine to form self-reactive TCRs of traditional TCR gene treatment are solved, and the safe and reliable new technology is provided for clinical anti-tumor T cell immunotherapy.

The invention provides a Ti plasmid aspergillus niger gene replacement expression vector and application thereof, and belongs to the technical field of molecular biology. The T-DNA (Triple helix Deoxyribose Nucleic Acid) region elements of the Ti plasmid aspergillus niger gene replacement expression vector are arranged in the following sequence: an aspergillus niger target gene promoter, a multiple cloning site, an aspergillus niger target gene terminator, an aspergillus nidulans 3-phosphoglyceraldehyde dehydrogenase gene promoter PgpdA, an aspergillus niger selection marker gene and an aspergillus niger target gene terminator. According to the invention, a target gene is integrated at the site of the aspergillus niger target gene through homologous recombination, and the target gene is regulated and controlled by a target gene promotor of high expression; therefore, the position effect of transgenosis is eliminated and the expression level is improved.

The invention discloses a method for detecting an enterovirus neutralizing antibody and a special recombinant virus for the method and provides a DNA (deoxyribonucleic acid) molecule. The DNA molecule is a recombinant DNA obtained by substituting coding genes of all structural proteins in cDNA (complementary DNA) corresponding to genome RNA (ribonucleic acid) of an enterovirus for report genes. An EV71 FY pseudovirus system is used for the method for detecting the neutralizing antibody, and since pseudoviruses subjected to monocyclic infection are adopted, the safety problem during using of live viruses is avoided. After multiple tests, results show that the pseudovirus system is the method for detecting the neutralizing antibody and is safe, sensitive, rapid, specific, simple, convenient and low in cost. Based on the advantages, the pseudovirus system is extremely suitable for rapid and massive neutralizing antibody detection tests, and has important application values for virus vaccine development and detection of hand-foot-and-mouth disease specificity neutralizing antibody level of individual patients and patient populations.

The present invention discloses transgenic monocot plants in which the plastid genome has been genetically engineered. The bioconfined genetically engineered monocot crops have transgene-free pollen grains which eliminate or dramatically reduce transgene flow. The present invention discloses plastid transgenesis technology having the additional advantages of the absence of gene silencing and position effect variation, the ability to express polycistronic messages from a single promoter, integration via a homologous recombination process that facilitates targeted gene replacement and precise transgene control, and sequestration of foreign proteins in the organelle which prevents adverse interaction with the cytoplasmic environment.

The invention discloses a traceless double-target genome editing system based on CRISPR/Cas9 and lambda-Red. The system is characterized in that 1, pKS9 has the molecular weight of 11,681 kb, is a thermo-sensitive plasmid, and contains cas9 and recX genes induced by tetracycline, lambda-Red integrity alpha-beta-gamma recombinase genes induced by arabinose, and resistance genes ampr; 2, pCSK has the molecular weight of 2,914 kb, and contains two sgRNA insertion sites for specific recognition of genomes, two spacer region gRNA sequences which are induced by rhamnose and used for plasmid elimination and resistance genes kanr; 3, when a homologous arm is 27 bp, the two-point mutation efficiency is both at 92% or above; when the homologous arm is 40 bp, the 1kb replacement efficiency of the twogenes is 60% or above, and when large-fragment gene replacement and point mutation are carried out simultaneously, the respective editing efficiency is not influenced.

The invention relates to a gateway inlet vector for the gateway technology, and in particular to a gateway inlet plasmid vector (pEn-L4*-PrbcS-*T-GFP-L3*). The vector comprises sequences L4 and L3, rubisco, a small subunit gene photo-inductive promoter (PrbcS), a chloroplast stroma positioning sequence (T*) and a green fluorescent protein (GFP) reporter gene, which are required for the LR recombination reaction in the gateway technology. A construction method and application of the vector are that: one plant expression vector connected in series with two target gene expression boxes can be quickly constructed by using the vector and another gateway inlet vector containing L1 and L2 sequences through the LR recombination reaction so as to realize conversion operation of two target genes through one conversion event. The expression of the target gene can be controlled with the PrbcS promoter in the vector and high-level expression of the target gene in the leaves of a plant can be realized by substituting the GFP gene in the vector with the target gene.

The invention provides a construction method and application of a humanized CCR2 gene modified animal model, and relates to the technical field of biology. According to the humanized CCR2 gene modified animal model constructed by the invention, the research progress of the fields related to the human CCR2 gene or protein can be accelerated; for example, the humanized CCR2 gene modified animal model is used for replacing a human to test a drug; and an effective model and a powerful tool are provided for a preclinical experiment of a CCR2 target drug. Preferably, a CRISPR/Cas9 gene editing technology is utilized in the invention; a mouse-derived Ccr2 gene is replaced with a human-derived CCR2 gene, so that a mouse model capable of interacting with an anti-human CCR2 antibody is constructed;compared with a common mouse, humanized transformation of key target molecules is achieved; and the mouse model can be used for screening and evaluating drugs for human CCR2 genes, and is a very idealpreclinical drug test model.

The invention belongs to the field of animal genetic engineering vaccines, and particularly relates to a fowl adenovirus serum type 4 (FAdV-4) recombinant virus for expressing fowl adenovirus serum type 8b (FAdV-8b) spike protein (Fiber) as well as a construction method and application of the fowl adenovirus serum type 4 (FAdV-4) recombinant virus. The recombinant virus is obtained by replacing a FAdV-4 Fiber1 gene with a Fiber gene of FAdV-8b, the recombinant virus can be used for preparing a bivalent vaccine for preventing and controlling chicken hepatitis-hydropericardium syndrome and/or chicken inclusion body hepatitis, and the bivalent vaccine prepared by utilizing the recombinant virus can achieve the effect of preventing two epidemic diseases at the same time by injecting a needle of vaccine. Therefore, the purpose of preventing and controlling two or more diseases by one needle can be achieved by inserting the FAdV-4 serving as a carrier into an exogenous gene.

The invention provides a construction method and application of an F gene replaced chimeric measles attenuated strain. Specifically, the invention provides the chimeric measles virus attenuated strain, and the attenuated strain is a measles virus rMV/F (H1a) with the preservation number of CCTCCNO: V202101. The invention also provides a vaccine composition containing the F gene substituted chimeric measles attenuated strain or the derivative virus strain thereof as an active ingredient and a preparation method of the vaccine composition. Compared with the existing measles vaccine strain in the market, the chimeric virus provided by the invention has stronger replication ability and better immunogenicity.

The present invention relates to a mutant Toxoplasma gondii which exhibits enhanced homologous recombination. The mutant of the present invention is a knockout in the KU80-dependent nonhomologous end-joining pathway which finds application in generating T. gondii gene knockouts and gene replacements for use in vaccine and drug development.

The invention belongs to the technical field of application of reverse genetics and gene replacement, and particularly relates to a chimeric Newcastle disease virus vaccine vector candidate strain capable of overcoming the influence of a maternal antibody of a Newcastle disease and a construction method of the chimeric Newcastle disease virus vaccine vector candidate strain. The candidate strain is avian paramyxovirus type-1 AI4-T4FHN, and the preservation number is CGMCC No:12987. An established reverse genetics manipulation platform for a Newcastle disease virus gene type-VII attenuated virus strain AI4 is utilized to replace the corresponding part of a genome of the strain AI4 through the sequence of an extracellular region of envelope proteins F and HN of an avian paramyxovirus type-2, and the recombinant virus AI4-T4FHN is obtained through saving. The cross reaction capacity of the avian paramyxovirus type-2 and the Newcastle disease virus are very weak, and the avian paramyxovirus type-2 is not pathogenic for a chicken. The chimeric virus has a high reproductive capacity similar to that of a female parent virus, and meanwhile, the chimeric virus is effectively replicated in the chicken body with the maternal antibody of the Newcastle disease at high level, and is a novel vaccine carrier.

The invention provides a method for preparing a recombinant virus. The method comprises the steps of using a virus vector as a matrix, introducing a bidirectional screening gene through Red/ET recombination, performing further recombination to replace the bidirectional screening gene with a target gene to obtain recombinant plasmid containing the target gene and a virus genome, and further performing salvation so as to obtain the recombinant virus. The method disclosed by the invention has the beneficial effects that the method is simple in steps, construction of the recombinant virus can be accurately and quickly realized, the recombinant virus which is successfully constructed can accommodate exogenous genes, advantages of multi-site orienteering inserting, traceless inserting and convenient mark screening can be realized, the recombinant virus which can express various antigen genes is formed finally, the recombinant virus is applied to vector vaccines, and the recombinant virus also has the advantages that the host range is wide, the production and preparation method is simple, the safety is good, various immune reactions are induced at the same time, and adjuvants do not needto be added.