844 results about "Recombinase" patented technology

This disclosure describe three related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of recombinase and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods have the advantage of not requiring thermocycling or thermophilic enzymes. Further, the improved processivity of the disclosed methods may allow amplification of DNA up to hundreds of megabases in length.

The invention relates to methods for inhibiting, cloning, modifying or labelling an endogenous DNA sequence using compositions comprising recombinases in combination with exogenous polynucleotides containing “anchoring” or “locking” sequences. The anchoring sequences serve to stabilize structures formed by the exogenous polynucleotides and the endogenous DNA. The stabilized structure thus can either serve to regulate gene transcription or replication, or can allow the endogenous sequences to be labelled or pulled out, i.e. cloned, or modified.

This disclosure describes related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of recombinase and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods have the advantage of not requiring thermocycling or thermophilic enzymes, thus offering easy and affordable implementation and portability relative to other amplification methods. Further RPA reactions using light and otherwise, methods to determine the nature of amplified species without a need for gel electrophoresis, methods to improve and optimize signal to noise ratios in RPA reactions, methods to optimize oligonucleotide primer function, methods to control carry-over contamination, and methods to employ sequence-specific third ‘specificity’ probes. Further described are novel properties and approaches for use of probes monitored by light in dynamic recombination environments.

Compositions, methods, strategies, kits, and systems for the supercharged protein-mediated delivery of functional effector proteins into cells in vivo, ex vivo, or in vitro are provided. Compositions, methods, strategies, kits, and systems for delivery of functional effector proteins using cationic lipids and cationic polymers are also provided. Functional effector proteins include, without limitation, transcriptional modulators (e.g., repressors or activators), recombinases, nucleases (e.g., RNA-programmable nucleases, such as Cas9 proteins; TALE nuclease, and zinc finger nucleases), deaminases, and other gene modifying / editing enzymes. Functional effector proteins include TALE effector proteins, e.g., TALE transcriptional activators and repressors, as well as TALE nucleases. Compositions, methods, strategies, and systems for the delivery of functional effector proteins into cells is useful for therapeutic and research purposes, including, but not limited to, the targeted manipulation of a gene associated with disease, the modulation of the expression level of a gene associated with disease, and the programming of cell fate.

A method for producing in vivo packaged recombinant adenovirus vectors is provided. The recombinant Ad vectors do not contain any Adenovirus genes and are therefore useful for gene therapy. The recombinant Adenovirus vectors are packaged in vivo using a helper virus which is itself very inefficiently packaged, providing a recombinant viral preparation with very little or no contamination with helper virus. In particular, the method makes use of a helper virus in which the packaging site can be easily excised in vivo by recombination mediated by a recombinase. The helper virus is also useful for the in vivo construction of new recombinant adenovirus vectors containing substitutions in the E1 or other adenoviral region.

The present invention relates to recombinantly modified enzymes, which exhibit increased NAD(H) affinity compared to a unmodified or wildtype enzyme, gene sequences or polynucleotides that code for the recombinantly modified enzymes, plasmids and microorganisms that contain these gene sequences; as well as, methods of making and methods of using the enzymes of the present invention.

The present invention features novel, diverse, hybrid and engineered recombinase enzymes, and the utility of such proteins with associated recombination factors for carrying out DNA amplification assays. The present invention also features different recombinase ‘systems’ having distinct biochemical activities in DNA amplification assays, and differing requirements for loading factors, single-stranded DNA binding proteins (SSBs), and the quantity of crowding agent employed.

The present invention provides compositions and methods for treatment of conditions and diseases associated with excessive or inappropriate noncancerous tissue growth. In certain embodiments of the invention the compositions and methods are used for treatment of benign prostatic hyperplasia. In certain embodiments of the invention the composition comprises a tissue-selective delivery vehicle. In certain embodiments of the invention the compositions comprise an expression vector that encodes a cytotoxic polypeptide, wherein expression of the cytotoxic polypeptide is under control of a prostate-specific regulatory element. In certain embodiments of the invention the compositions comprise an expression vector in which expression of a recombinase is under control of a prostate-specific regulatory element, and a recombination event mediated by the recombinase is required for expression of the cytotoxic polypeptide.

A gene activation / inactivation and site-specific integration system has been developed for mammalian cells. The invention system is based on the recombination of transfected sequences by FLP, a recombinase derived from Saccharomyces. In several cell lines, FLP has been shown to rapidly and precisely recombine copies of its specific target sequence. For example, a chromosomally integrated, silent β-galactosidase reporter gene was activated for expression by FLP-mediated removal of intervening sequences to generate clones of marked cells. Alternatively, the reverse reaction can be used to target transfected DNA to specific chromosomal sites. These results demonstrate that FLP can be used, for example, to mosaically activate or inactivate transgenes for a variety of therapeutic purposes, as well as for analysis of vertebrate development. The FLP recombination system of the present invention can be incorporated in transgenic, non-human mammals to achieve site-specific integration of transgenes, to construct functional genes or to disrupt existing genes.

Methods are provided for producing an expression vector. In the subject methods, donor and acceptor vectors are combined in the presence of a recombinase to produce an expression vector that includes a first and second recombinase recognition site oriented in the same direction, wherein the first and second recombination sites are able to recombine with each other. In the subject methods, one of the donor and acceptor vectors includes a single recombinase recognition site while the other includes two recombinase recognition sites. Also provided are compositions for use in practicing the subject methods, including the donor and acceptor vectors themselves, as well as systems and kits that include the same. The subject invention finds use in a variety of different applications, including the transfer or cloning of a nucleic acid of interest from a first vector into one or more expression vectors, etc.

This disclosure describes related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of recombinase and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods have the advantage of not requiring thermocycling or thermophilic enzymes, thus offering easy and affordable implementation and portability relative to other amplification methods. Further RPA reactions using light and otherwise, methods to determine the nature of amplified species without a need for gel electrophoresis, methods to improve and optimize signal to noise ratios in RPA reactions, methods to optimize oligonucleotide primer function, methods to control carry-over contamination, and methods to employ sequence-specific third ‘specificity’ probes. Further described are novel properties and approaches for use of probes monitored by light in dynamic recombination environments.

Compositions and methods are provided for the introduction and the regulated expression of genes in plants. Compositions include promoter constructs that provide a level of activity useful for the regulated expression of site-specific recombinases, while avoiding premature excision. Further provided are isolated polynucleotides encoding novel babyboom polypeptides, expression cassettes, and plants comprising the same. Methods for the introduction of genes into plants are provided, including methods for plastid transformation and methods for the transformation of tissues from mature seeds and leaves.

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 relates to a rapid construction method of a conditional gene knockout animal model, which is implemented by using a CRISPR / Cas9 technology. The specific method can be implemented as follows: designing a gRNA target spot sequence, for a to-be-knocked-out gene of an animal, of a CRISPR / Cas9 system, designing a corresponding promoter, constructing a plasmid, thereby implementing conditional gene knockout. According to the invention, the rapid, efficient, simple, feasible, economic, efficient, and wide-applicable-scope construction of a conditional gene knockout animal model can be realized, and the defects existing in traditional methods (specific recombination enzyme system Cre-LoxP) can be overcome.

Methods for the targeted integration of nucleotide sequences into a plant are provided. Transfer cassettes comprising nucleotide sequences of interest flanked by non-identical recombination sites are used to transform a plant comprising a target site. The target site contains at least a set of non-identical recombination sites corresponding to those on the transfer cassette. Exchange of the nucleotide sequences flanked by the recombination sites is effected by a recombinase.

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 novel carbonyl reductase, a gene, a mutant thereof, a recombinant expression vector containing the gene and the mutant, a recombinant expression transformant, a recombinase preparation method, and applications of the carbonyl reductase and recombinase to preparation of active chiral alcohols with a chiral carbonyl compound before asymmetrical reduction. The carbonyl reductase is derived from candida glabrata, is applied to preparation of a plurality of optically-active chiral alcohols such as (R)-chloromandelic acid methyl ester, (R)-2-hydroxy-4-phenyl ethyl butyrate, (R)-4-chlorin-3-phenyl ethyl butyrate and the like. Compared with other preparation methods, a product prepared through the method has high concentration, does not require additionally or slightly adding any expensive coenzyme, has high optical purity, and has the advantages of mild reaction conditions, easiness and convenience for operating, easiness for amplifying and the like, and has a good industrial application prospect in the production of clopidogrel, L-carnitine and perindopril antihypertensive medicinal intermediates.

Methods for the targeted integration of nucleotide sequences into a plant are provided. Transfer cassettes comprising nucleotide sequences of interest flanked by non-identical recombination sites are used to transform a plant comprising a target site. The target site contains at least a set of non-identical recombination sites corresponding to those on the transfer cassette. Exchange of the nucleotide sequences flanked by the recombination sites is effected by a recombinase.

Disclosed are variants of Cre recombinase that have broadened specificity for the site of recombination. Specifically, the disclosed variants mediate recombination between sequences other than the loxP sequence and other lox site sequences on which wild type Cre recombinase is active. In general, the disclosed Cre variants mediate efficient recombination between lox sites that wild type Cre can act on (referred to as wild type lox sites), between variant lox sites not efficiently utilized by wild type Cre (referred to as variant lox sites), and between a wild type lox site and a variant lox site. Also disclosed are methods or recombining nucleic acids using the disclosed Cre variants. For example, the disclosed Cre variants can be used in any method or technique where Cre recombinase (or other, similar recombinases such as FLP) can be used. In addition, the disclosed Cre variants allow different alternative recombinations to be performed since the Cre variants allow much more efficient recombination between wild type lox sites and variant lox sites. Control of such alternative recombination can be used to accomplish more sophisticated sequential recombinations to achieve results not possible with wild type Cre recombinase.

The present invention provides methods of producing transgenic livestock animals. The methods generally involve first introducing a nucleoprotein made up of nucleic acid and a recombinase into a totipotent or pluripotent cell to produce a recombinant totipotent or pluripotent cell and then growing the recombinant totipotent or pluripotent cell to produce the transgenic livestock animal. The invention further provides kits for use in generating transgenic non-human animals of the invention.

The invention relates to a construction method of a homologous repair vector based on a CRISPR / Cas9 system, and belongs to the technical field of genetic engineering. The construction method comprisesthe following steps: with a plasmid PCBC and wild-type arabidopsis genome as templates, amplifying a target band AS-gRNA containing a target sequence and an AS homologous repair template segment by virtue of PCR, implementing electrophoresis and gel cutting, and recovering the target band; implementing enzyme digestion on a plasmid PHDE-mCH by virtue of Bsa1; assembling and linking a PHDE-mCh vector, which undergoes complete enzyme digestion, with the ASgRNA by virtue of homologous recombinase, so that a recombinant plasmid PHDE-ASgRNA is formed, implementing transformation and sequencing identification, then linking the PHDE-ASgRNA plasmid, which is correct in sequencing and is subjected to enzyme digestion by virtue of EcoR1, with AS homologous repair template segment homologous recombinase, and implementing transformation and sequencing identification, so that the PHDE-ASgRNA-AS homologous repair vector is constructed. According to the technique provided by the invention, the method for constructing the CRISPR / Cas9 system which has a site-specific editing function on target biology genome is actually implemented; the vector is constructed just by conducting PCR by one step, sothat the method is simple and easy to implement; and the method, which is unnecessary to purify or recover a digestion vector and a PCR product, is high in assembly efficiency.

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 invention relates to the clone and the expression of alpha-cyclodextrin glycosyltransferase (alpha-CGTase), which belong to the field of enzyme gene engineering and enzyme engineering. The cgt gene expression method comprises the following steps: obtaining cgt gene SEQ ID NO:1 from total DNA of Peanibacillus macerans JFB05-01; selecting plasmid pET20b(+) as the expression vector of cgt gene; and selecting E.coli BL21(DE3) as the expression host to achieve high-efficiency extracellular expression of the cgt gene, wherein the cgt gene has 2,061 nucleotides and 687 coded amino acids; prokaryotic expression plasmid is constructed; and alpha-CGTase is expressed by transformed E.coli. Recombinase has cyclization activity and can transform starch and relevant matrix into cyclodextrin. The recombined alpha-CGTase has an optimal temperature of 40 to 45 DEG C and an optimal pH value of 5.5, and has higher thermal stability below 40 DEG C and poor thermal stability above 50 DEG C. The alpha-CGTase meets the requirement for industrial application such as food and medicines, and can be used for industrial production of alpha-cyclodextrin and beta-cyclodextrin.

The invention relates to a carbonyl reductase CgKR1 mutant, a coding gene of the mutant, a recombinant expression vector containing the gene of the carbonyl reductase mutant, a recombinant expression transformant, a recombinase, a preparation method of the recombinase, and an application of the carbonyl reductase mutant to asymmetric reduction of ketonic ester for preparation of optically pure chiral hydroxyl ester, such as catalysis of o-cyano methyl phenylglyoxylate for asymmetric reduction to prepare (R)-o-chloro mandelic acid methyl ester. Compared with wild enzymes, the carbonyl reductase mutant has the advantages that the thermal stability is substantially improved, and the catalytic activity of part of the mutant to the o-chlorobenzoic acid formyl methyl ester is also obviously improved. The multiple mutants can be applied to catalysis of the ketonic ester for asymmetric reduction to prepare the optical purely-chiral hydroxyl ester, such as catalysis of the o-cyano methyl phenylglyoxylate for asymmetric reduction to prepare the optically pure (R)-o-chloro mandelic acid methyl ester. The carbonyl reductase mutants have the very good industrial application prospect.

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.

A real-time isothermal recombinase-polymerase amplification detection kit for African swine fever viruses is disclosed. A forward primer sequence for detecting the African swine fever viruses through a method provided by the kit is shown as SEQ ID NO:1, a reverse primer sequence is shown as SEQ ID NO:2 and a probe sequence is shown as SEQ ID NO:3. A real-time isothermal recombinase-polymerase amplification method provided by the invention for ASFV detection is simple and convenient in operation, rapid in reaction and low in detection cost, can be used for ASFV detection in a laboratory and on site, particularly ASFV detection in quarantine ports, airports and epidemic disease outbreak sites, and provides a novel and reliable technique support for ASF control in China.

A helper dependent adenoviral vector system is provided. The subject helper dependent adenoviral vector system is made up of: (1) a "gutless" adenoviral vector that include cis-acting human stuffer DNA that provides for in vivo long term, high level expression of a coding sequence present on the vector; (2) an adenoviral helper vector that is characterized by having an adenoviral genome region flanked by recombinase recognition sites, where the helper vectors further include a non-mammalian endonuclease recognition site positioned outside of the adenoviral genome region; and (3) a mammalian cell that expresses the corresponding recombinase and endonuclease, as well as the adenoviral preterminal and polymerase proteins. Also provided are methods of using the subject systems to produce virions having the subject helper dependent adenoviral vectors encapsulated in an adenoviral capsid. In addition, kits for use in practicing the subject methods are provided.

A gene targeting method for use in a host organism whereby the host organism is transfected with an ends-out donor construct. Further transfecting the host organism with two transgenes expressing endonuclease and recombinase enzymes. The endonuclease and recombinase enzymes are used so that homologous recombination occurs between the DNA segment of the donor construct and a selected gene of the host organism, thus forming a host with containing the recombinogenic donor. The progeny of the host organism, which include the recombinogenic donor are then selected.