141 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 present invention is based on the discovery that a high titer reassortant influenza virus is produced in mammalian cell culture by replacing the NS gene of the A / PuertoRico / 3 / 24 master strain with the NS gene of the A / England / 1 / 53 strain. The invention provides influenza viruses and vaccines generated in mammalian cells as well as methods for producing such. The invention further provides an influenza virus master strain and kits for generating reassortant influenza viruses in mammalian cell culture and methods of making and using the master strain.

To provide DNA comprising mutant FRT sequence which causes recombination reaction between two mutant FRT sequences having an identical sequence to each other but does not cause recombination reaction with a wild-type FRT sequence, in the presence of FLP recombinase; and a method for performing high-efficiency, gene insertion or gene replacement. A DNA comprising a mutant FRT sequence. A DNA comprising a mutant FRT sequence possessing (A) causing no specific DNA recombination reaction with wild type FRT, even if FLP recombinase is present, and (B) causing specific DNA recombination reaction with another mutant FRT sequence having an identical sequence thereto in the presence of recombinase FLP; gene replacement method using the DNA in the presence of recombinase FLP; and a specific DNA recombination method, characterized in that a specific DNA recombination reaction is carried out by using two mutant FRT sequences in the presence of recombinase FLP.

The present invention relates to the use of tumor suppressor genes in combination with a DNA damaging agent or factor for use in killing cells, and in particular cancerous cells. A tumor suppressor gene, p53, was delivered via a recombinant adenovirus-mediated gene transfer both in vitro and in vivo, in combination with a chemotherapeutic agent. Treated cells underwent apoptosis with specific DNA fragmentation. Direct injection of the p53-adenovirus construct into tumors subcutaneously, followed by intraperitoneal administration of a DNA damaging agent, cisplatin, induced massive apoptotic destruction of the tumors. The invention also provides for the clinical application of a regimen combining gene replacement using replication-deficient wild-type p53 adenovirus and DNA-damaging drugs for treatment of human cancer.

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.

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 present invention discloses a method for improving Bombyx mori sec-linked balanced lethal line by using backcross process. Which is characterized by that it adopts three processes of crossing, selfing and backcross of balanced lethal line and conventional quality variety to form haematodes circulation, and utilizes said haematodes circulation to progressively substitute the gene of quality variety into the Bombyx mori sec-linked balanced lethal line, at the same time adopts marker gene selection measure to raise its economic trait on the premise of that the sex-controlling gene of balanced lethal line is not lost to reach the economic trait level of quality variety. Said invention is simple in operation method, and easy to popularize.

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 provides Salmonella typimurium S496. The Salmonella typimurium S496 is characterized by lacking an rfbB gene and an rffG gene; a pagL gene is replaced by an arabinose-regulatory-sequence-containing rfbB gene with a changed SD sequence and a changed initiation codon, and the nucleotide sequence of the arabinose-regulatory-sequence-containing rfbB gene with the changed SD sequence and the changed initiation codon is shown as SEQ ID No.1; the classification of the attenuation strain is named as the Salmonella typimurium S496, the Salmonella typimurium S496 is collected in the China Center For Type Culture Collection (CCTCC), the collection number is CCTCC M2015562, and the collection time is September 21st, 2015. The invention further provides a method for constructing the Salmonella typimurium S496. Meanwhile, the invention provides an application of the strain or the attenuation strain constructed with the construction method in vaccine.

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 molecular marker vaccine strain for Brucella melitensis and application of the molecular marker vaccine strain. Encoding genes of bp26 proteins of the Brucella melitensis MB6 are replaced by encoding genes of BLS proteins and encoding genes of L7 / L12 proteins, and encoding genes of wboA proteins of the Brucella melitensis are inactivated, so that the molecular marker vaccine strain for the Brucella melitensis can be obtained. The molecular marker vaccine strain and the application have the advantages that as proved by experiments, excellent immune protection effects can be realized by the Brucella melitensis for Brucellosis, the molecular marker vaccine strain is low in virulence and high in safety and can be used for immunoprophylaxis for the Brucellosis of sheep, cows and the like, immunized animals can be differentiated from wild-strain-infected animals by the aid of immunological techniques, and accordingly the molecular marker vaccine strain and the application have important significances on monitoring, diagnosing, purifying and controlling the Brucellosis and have wide application value.

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 discloses a pidemic encephalitis B / dengue chimeric virus with an epidemic encephalitis B virus attenuated strain as a gene framework and application of the epidemic encephalitis B / dengue chimeric virus. The invention provides a deoxyribonucleic acid (DNA) molecule, and the DNA molecule is recombinant DNA which is obtained by replacing an encoding gene of protein A in complementary DNA (cDNA) corresponding to genome ribonucleic acid (RNA) of an epidemic encephalitis B virus by using an encoding gene of protein B; the protein A consists of an amino acid sequence shown as a sequence 1 in a sequence table; and the protein B consists of an amino acid sequence shown as a sequence 2 in the sequence table. The pidemic encephalitis B / dengue chimeric virus with the epidemic encephalitis B virus attenuated strain as the gene framework is a recombinant virus containing the DNA molecule. The pidemic encephalitis B / dengue type II chimeric virus also has a good application prospect when taken as a dengue vaccine and used for preventing dengue virus infection.

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 provides an O-type foot and mouth disease virus strain with improved replication titer as well as a construction method and application thereof, and belongs to the technical field of biological products. Through a reverse genetic manipulation technology of the foot and mouth disease virus, a G-H ring gene of the foot and mouth disease virus O / NXYCh / CHA / 2018 strain is further used for replacing a corresponding gene on a recombinant virus skeleton of a main immune gene of the chimeric foot and mouth disease virus O / XJ / CHA / 2017 strain, the time of causing 100% infected cell lesion by the constructed recombinant virus rHN / XJ / NXGH is remarkably shortened to 12 h, and the time of causing 100% infected cell lesion by the recombinant virus rHN / XJ / NXGH is remarkably shortened to 12 h. And the replication titer of infected cells for 12 hours is improved by more than 5 times. The recombinant virus is continuous in passage and good in hereditary stability, and replacement of the G-H ring does not affect immunogenicity of the vaccine. Therefore, the recombinant foot-and-mouth disease virus strain rHN / XJ / NXGH provided by the invention has the potential to be used as a vaccine candidate strain for effectively preventing and controlling the O-type foot-and-mouth disease in China.

The invention discloses a method for increasing S-adenosyl-L-methionine yield by saccharomyces cerevisiae genetic engineering. The method includes: replacing a GLC3 (glycogen branching enzyme gene) allelic gene on a chromosome of a saccharomyces cerevisiae strain with a G418 resistance gene according to a gene replacement method, and using a spore isolation method for obtaining a haploid with the gene GLC3 replaced only, so that a homozygote with gene GLC3 mutated is obtained. According to fermentation in 10L and 500L fermentation tanks, yields of ademetionine produced with mutant strains reach 7.93g / L and 8.35g / L and are increased by 15.1% and 24.7% respectively as compared with that of ademetionine produced with original strains.

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.

Methods and compositions for treating disease using human subgroup B adenovirus, vectors derived from such viruses, including expression vector systems in which one or more subgroup B adenoviral genes are replaced by a foreign gene.

The present invention relates to the use of tumor suppressor genes in combination with a DNA damaging agent or factor for use in killing cells, and in particular cancerous cells. A tumor suppressor gene, p53, was delivered via a recombinant adenovirus-mediated gene transfer both in vitro and in vivo, in combination with a chemotherapeutic agent. Treated cells underwent apoptosis with specific DNA fragmentation. Direct injection of the p53-adenovirus construct into tumors subcutaneously, followed by intraperitoneal administration of a DNA damaging agent, cisplatin, induced massive apoptotic destruction of the tumors. The invention also provides for the clinical application of a regimen combining gene replacement using replication-deficient wild-type p53 adenovirus and DNA-damaging drugs for treatment of human cancer.

The invention belongs to the field of biology, and discloses a recombinant plasmid pBR322-FDHN3-S1. The sequence of the recombinant plasmid pBR322-FDHN3-S1 is shown as a sequence table SEQ ID NO: 1. The recombinant plasmid is based on a VII subtype NDV strain DHN3 strain with strong toxicity, an F gene of a Lasota attenuated strain is used for replacing an F gene of the DHN3 strain, an S1 gene is inserted, and the recombinant plasmid has immunogenicity for II-type and VII-type Newcastle diseases and IBV. Meanwhile, the invention also discloses a recombinant virus and a high-titer culture method for the virus.

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 aims at providing a two-way starting plant expression vector system of double recombination sites. The two-way starting plant expression vector system is composed of a two-way expressing plant expression vector pRGFL of double recombination sites and two cloning vectors p-FRT and p-loxp with specific recombination sites respectively. The two-way expressing plant expression vector pRGFL of the double recombination sites provided by the invention is a transformed plant expression vector containing specific DNA molecules. The two cloning vectors with the specific recombination sites respectively provided by the invention are respectively inserted into the recombination vector containing the specific DNA molecules at multiple cloning sites of a starting vector. The cloning vector p-FRT and the cloning vector p-loxp provided by the invention form a vector with a T tail end or a flat tail end after being digested by XcmI enzyme or EcorV enzyme, and can be directly connected with a PCR product of a target gene to achieve the cloning target. The target genes on the cloning vector p-FRT and the cloning vector p-loxp can respectively replace fluorescence protein reporter genes on the two-way expressing plant expression vector of the double recombination sites through specific site combination, and thus the target genes are transferred to the plant expression vector; only enzyme medium needs to be recombined in the process; and other operations are not required, so that the two-way starting plant expression vector system is simple and feasible.

Methods and means are provided for the exact exchange in eukaryotic cells, such as plant cells, of a target DNA sequence for a DNA sequence of interest through homologous recombination, whereby the selectable or screenable marker used during the homologous recombination phase for temporal selection of the gene replacement events can subsequently be removed without leaving a foot-print employing a method for the removal of a selected DNA flanked by two nucleotide sequences in direct repeats.