110 results about "Tyrosine recombinase" patented technology

A modified P. aeruginosa type III secretion system has been developed that efficiently delivers selected proteins into a host cell. In one example, a functional nuclear Cre Recombinase is injected into embryonic stem (ES) cells and can be used to induce pluripotent stem (iPS) cells. This method of in vitro lineage directed differentiation prevents insertional mutagenesis and provides a route to selected stem cell renewal and cell-based therapies.

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 invention relates to the technical field of high throughput sequencing, and discloses a method for establishing a DNA (Deoxyribose Nucleic Acid) library based on an illumina sequencing platform. The method comprises the following steps: (1) randomly interrupting the genome DNA; (2) filling the tail ends, and adding biotin labeled cyclizing connectors at two ends; (3) separating DNA fragments, wherein the sequence of the step (2) and the step (3) can be changed;; (4) cyclizing, and removing uncyclized DNA fragments; (5) randomly interrupting cyclized DNA; (6) separating biotin labeled DNA fragments; (7) filling the tail ends of the biotin labeled DNA fragments; (8) adding an A alkali group; (9) connecting with the sequencing connectors; (10) performing PCR amplification and enrichment, thereby obtaining the sequencing library. The method adopts a Cre recombinase system to cyclize the DNA fragments, the cyclizing only takes 1 hour, the time for establishing the library is greatly shortened, the large-fragment DNA library is successfully established, and splicing of genomes in sequencing can meet the standard of a fine diagram or accomplished diagram.

Provided is a method of preparing an induced pluripotent stem cell (iPS cell) deprived of a reprogramming gene, including providing an iPS cell having an expression vector wherein a loxP sequence is placed on each of the 5′ and 3′ sides of the reprogramming gene or a vector component necessary for the replication of the reprogramming gene in the same orientation, and treating the IPS cell with Cre recombinase. Also provided are an iPS cell deprived of a reprogramming gene, as obtained by the method, and a use of the iPS cell as a cell source for producing somatic cells.

The present invention provides a transgene report pig detecting Cre site special recombinase active and a culturing methodThe method includes steps as follows: building LoxP site anchoring end terminal, receiving a target carrier of report gene, when Cre recombinase exist, deleting the end terminal anchored by Loxp, and expressing the received report gene, accordingly, monitoring Cre recombinase activity; filter out a fibroblast which can be special detected Cre activity through cell transfection and cell filtering technique; using body nucleus transplantation technique for processing cloning pig. The method is used for monitoring Cre recombinase activity in body, provides a monitoring tool for establishing human disease model, solves problems of immature technique for transgene cloning pig and low efficiency.

The invention relates to a method for conditional knockout of Lox13 gene of a mouse and an application. The method comprises the steps that loxP loca of a Cre recombinase targeting sequence are added to the two ends of a key exon of an Lox13 gene; a conditional gene knockout carrier is constructed, and transfected with an embryonic stem cell (ESC); microinjection is conducted for making a floxed mouse capable of being used for conditional gene knockout; the floxed mouse is mated with a Cre mouse with corresponding tissue specificity; and the mouse of which an Lox13 gene can be knocked out at a specific tissue is obtained. The mouse obtained by the method serves as a model mouse for morbidity research on a cleft palate disease, so that the functions of the Lox13 gene in the tissue and the effects of the Lox13 gene in an embryonic development regulation mechanism can be illustrated in details. In addition, the mouse can serve as an animal model for researching the functions of the Lox13 gene, and has important application values.

The invention relates to a construction method of an AURKA-CKO1-N condition gene knockout mouse model. A recombination targeting carrier is constructed, embryonic stem cell are transfected after linearization, embryonic stem cell with homologous recombination are screened for clone, a donor mice blastula is injected after amplification to produce a chimera mouse; the chimera mouse copulates with an flp mouse to obtain a male F1 generation of mouse; and the male F1 generation of mouse copulates with a mouse whose tissues include specific Cre recombinase, and the AURKA-CKO1-N condition gene knockout mouse model is obtained in the next generation. According to the AURKA-CKO1-N condition gene knockout mouse model, an AURKA gene loses functions in specific tissues and cells, and an ideal modelis provided for research on an action mechanism of Aurora-A in tumorigenesis development.

The present provides reversibly immortalized RPTECs and methods for making and utilizing these cells. Specifically, the present invention provides a method of reversibly immortalizing RPTECs by introducing a first vector containing a human telomerase catalytic subunit (hTERT) gene flanked by loxP sites and a second vector containing an SV40 T antigen (Tag) gene flanked by loxP sites. Immortalization can be reversed by introduction of a third vector containing a Cre recombinase or Cre variant gene. The reversibly immortalized RPTECs generated by this method may be used for a variety of applications, including screening of test agents for the ability to modulate renal toxicity or incorporation into devices designed to mimic the activity of the renal proximal tubules.

Disclosed are materials and methods for creating customizable traits in animals. In the demonstration of the principle of the subject invention, a keratin-14 specific promoter is used with, red fluorescent protein in the loxp cassette, dominant black (ΔG23) beta defensin 103 in the pigment cassette, and an SV40 (with intron) polyadenylation sequence. When Cre recombinase (or HTNCre) is applied to the animal's skin in a carrier base (e.g., lipid bilayers), fur is permanently genetically modified to turn black in the shape in which it was applied.

The invention relates to the technical field of animal models and in particular relates to a method for building a human psoriasis mouse model and application of the model to drug screening. The method adopts a conditional gene targeting technology and comprises the following steps: creating a marked mouse with homologous loxP sites on the two sides of an IKK2 gene through a homologous recombination process, hybridizing the marked mouse with a mouse with Cre recombinase expression under the control of a tissue-specific promoter SM22, and screening to obtain the human psoriasis mouse model. The human psoriasis mouse model has the advantages that the building method is simple, convenient and economic, and the psoriasis expression of the human psoriasis mouse model is highly similar to that of a human body; moreover, the animal model is convenient to use and has market values and medical application prospects.

The invention provides a transgene pig for preparing an animal model for human diseases and a culturing method thereof. An inducible expression Cre recombinase expression vector pET28a-Mxl-Cre-BGHpolyA-FRT2neoR is constructed by a genetic engineering method, and can express Cre recombinase after the expression vector is induced by interferon inducer poly IC (poly I:C) so as to be used for knocking out a target gene; porcine mechanocyte capable of inducibly expressing the Cre recmbinase can be screened through technology of cell transfection and cell screening; and the pig is cloned by the technology of somatic cell nuclear transplantation. The cloned pig can be used for preparing the animal model for knocking out genes of human diseases.

Disclosed are materials and methods for creating customizable traits in animals. In the demonstration of the principle of the subject invention, a keratin-14 specific promoter is used with red fluorescent protein in the loxp cassette, dominant black (ΔG23) beta defensin 103 in the pigment cassette, and an SV40 (with intron) polyadenylation sequence. When Cre recombinase (or HTNCre) is applied to the animal's skin in a carrier base (e.g., lipid bilayers), fur is permanently genetically modified to turn black in the shape in which the HTNCre was applied.

The invention discloses a Cre fusion protein functional test cell and a modified Cre fusion protein. The Cre fusion protein functional test cell takes an HEK (human embryonic kidney) 293 cell as a host cell, a pAcmy (African cassava mosaic virus)-stop-EGFP (enhanced green fluorescent protein) carrier and phi C31 integrase are used for co-transfection of the host cell. Through the action of the phi C31 integrase, the carrier which carries an LoxP (locus of X-over P1) element having a Cre recombinase detection function is integrated to a pseudo attP (attachment P) site of an HEK 293 cell line in a single copying manner, so a stable and reliable cell model is provided for researching the Cre fusion protein membrane penetrating efficiency and biological activity. The Cre fusion protein HNTC (hemagglutinin-neuraminidase cytotoxic T) which is modified by TAT (transactivator) and NLS (nuclear localization signal) polypeptides has high membrane penetrating efficiency and good biological activity, can be effectively used as a tool for genetic manipulation at a mammalian cell chromosome level.

Some aspects of this disclosure provide a fusion protein comprising a guide nucleotide sequence-programmable DNA binding protein domain (e.g., a nuclease-inactive variant of Cas9 such as dCas9), an optional linker, and a recombinase catalytic domain (e.g., a tyrosine recombinase catalytic domain or a serine recombinase catalytic domain such as a Gin recombinase catalytic domain). This fusion protein can recombine DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. The instant disclosure represents a step toward programmable, scarless genome editing inunmodified cells that is independent of endogenous cellular machinery or cell state.

Disclosed are materials and methods for creating customizable traits in animals. In the demonstration of the principle of the subject invention, a keratin-14 specific promoter is used with, red fluorescent protein in the loxp cassette, dominant black (ΔG23) beta defensin 103 in the pigment cassette, and an SV40 (with intron) polyadenylation sequence. When Cre recombinase (or HTNCre) is applied to the animal's skin in a carrier base (e.g., lipid bilayers), fur is permanently genetically modified to turn black in the shape in which it was applied.

The invention discloses a Cre recombinase recombination gene and a Cre / loxP-mediated transgenic safe plant expression vector, relates to a Cre recombinase recombination gene and a plant expression vector and solves the problems of the traditional method for removing marker genes in transgenic plants by the traditional Cre / loxP locus specific recombination system has the problem that the deleting efficiency is reduced because the Cre / loxP locus specific recombination system has the reversibility. The crein gene sequence is as shown in SEQ ID NO:1. The vector contains a maker gene bar, a crein gene, a heat shock promoter hsp and a forming type promoter CaMV35S. The invention can improve the safety of the transgenic plants.

Belonging to the field of biotechnologies, the invention discloses an expression vector containing regulatably expressible cre recombinase gene and reporter gene LacZ, a construction method and application thereof. The expression vector provided by the invention is pBI-3-LacZ-cre, and the expression vector contains the lacZ reporter gene, the Cre recombinase gene and a bidirectional promoter tetO element. According to the invention, the expression vector pBI-3-LacZ-cre is introduced into a mouse fallopian tube to conduct pregnancy so as to obtain a transgenic mouse. The transgenic mouse is hybridized with two applied model animals, i.e. an rTALAP-1 mouse able to induce liver specific expression of rtTA molecules and an HBV Tg (Transgenic) mouse able to regulatably express HBV genome, and then after repeated mating, a liver inducibly expressible Tet-on and Cre / loxP combined dual-control strategy triple-transgenic mouse new model can be obtained finally. The expression vector can be effectively applied to screening and evaluation of HBV (hepatitis B virus) inhibitors or drugs, research of HBV pathogenic mechanism, host immune response after HBV infection and other key issues.

The invention discloses a construction and an application of a mouse model with conditional knockout of an MACF1 gene in osteoblast and is used for solving the technical problem that the practicality is poor in the prior art. The technical scheme is that the mouse model (Sp7-Cre; MACF1f1 / f1) with conditional knockout of the MACF1 gene in the osteoblast is obtained by means of a Cre-Loxp system by mating a MACF1f1 / f1 mouse with a Sp7-Cre transgenic mouse, osteoblast of which specifically expressed Cre recombinase. The bone forming ability of the mouse with conditional knockout of the MACF1 gene in the osteoblast is reduced. The model can be used for researching the function of the MACF1 gene in the osteoblast and the effect thereof in a bone forming process, and meanwhile, can be used as an animal model for researching pathogenesis / generation mechanism of osteoporosis or other bone formation related diseases, and is used for screening drugs for preventing, treating or improving the diseases.

The invention discloses a lentiviral expression vector pLOX-CMV-E / P and construction and application thereof. Multiple cloning site which is inserted into the downstream part of a CMV promoter of the vector can be recognized by 7 restriction endonucleases, including SpeI, BclI, EcoRI, Paci, XhoI, SalI and BamHI. The application of the vector comprises the following steps: inserting a target gene to the multiple cloning site to realize the stable expression of the target gene; using Puromycin resistance gene and EGFP which is driven by EF1alpha promoter to perform fluorescence tracing and drug resistance screening; based on LoxP site inserted in 3'LTR region and the transcription and reverse transcription characteristic of lentivirus, realizing effective deleting of an exogenous gene and a reporter gene under the condition of Cre recombinase expression. The vector disclosed by the invention can realize the package of the lentiviral particles by commonly transfecting 293T cell together with pCMVR8.74 and PMD2.G vector.

The invention discloses a mouse model with the IRTKS gene in the liver specifically eliminated as well as construction and applications of the mouse model. Through homologous recombination, a 2.245-kb frt-neo-frt-loxp-Flox-loxp sequence replaces a sequence containing exon2 in the mouse IRTKS gene, and thus the chimeric mouse with IRTKS<flox / +> is obtained; by utilizing the Cre recombinase, the obtained chimeric mouse mates with the mouse Alb-Cre with specific expression of Cre of the liver, and finally, the mouse model with IRTKS gene in the liver specifically eliminated is obtained. The mouse model with the IRTKS gene eliminated constructed by adopting the method provided the invention can be used for researching diabetes mellitus; through the body weight, fasting blood-glucose, sugar tolerance and clinicopathologic analysis, the result shows that the features conform to the human diabetes mellitus morbidity features, and thus a good animal model is provided for researching the diabetes mellitus morbidity mechanism and evaluating and screening treatment medicines.

Disclosed are materials and methods for creating customizable traits in animals. In the demonstration of the principle of the subject invention, a keratin-14 specific promoter is used with red fluorescent protein in the loxp cassette, dominant black ([Delta]G23) beta defensin 103 in the pigment cassette, and an SV40 (with intron) polyadenylation sequence. When Cre recombinase (or HTNCre) is applied to the animal's skin in a carrier base (e.g., lipid bilayers), fur is permanently genetically modified to tum black in the shape in which theHTNCre was applied.

The invention discloses a HBD3 mammary gland specific expression vector and a constructed recombinant cell. The vector comprises a HBD3 gene with a signal transduction peptide, and CSN5 and CSN3 are set upstream and downstream of the HBD3 gene respectively for serving as regulating and controlling elements. The host cell of the recombinant cell is a bovine fetal fibroblast cell, an exogenous expression vector pARNG-HBD3 is integrated to a pseudo attP locus under the action of phiC31 integrase, positive cloning is realized through medicament screening, and an antibiotic screening marker between two homodromous LoxP sequences on the vector is removed through Cre recombinase treatment. A HBD3-containing bovine fetal fibroblast cell from which an antibiotic screening marker is removed is taken as a nuclear donor cell of a transgenic cloning embryo through nucleus transplantation, gene cloning embryos are obtained through SCNT (Somatic Cell Nuclear Transfer), and a milk cow which is free from a HBD3 transgenic gene of the antibiotic screening marker can be produced by transferring these embryos into a receptor cow.

The invention discloses a yeast genome editing carrier as well as a construction method and applications of the yeast genome editing carrier. According to the technical scheme, a gRNA coding plasmid Pis adopted as a starting carrier, LoxP sequences of 34bp in the same direction are introduced at two ends of the replication initial point Ori of the carrier, a Cre recombinase gene expression box isintroduced at the skeleton position of the plasmid P, and thus the yeast genome editing carrier is obtained. The carrier is applied in gene editing adopting CRISPR / Cas9 as the medium, and the efficient and multi-round editing for the saccharomyces cerevisiae genome can be realized.

Traditional Cre transgene has difficultly in carrying out tissue-specific expression. The invention carries out innovation based on a double transgenic technique and a Flp-Frt technique. The invention is characterized in that: firstly, an STOP original part is added in front of a translation initiation codon of Cre to inhibit the expression of downstream Cre, and two Frt sequences are separately added to the upstream and downstream of the STOP original part; then the STOP original part between the two Frt sequences can be specifically deleted by utilizing Flp recombinase, thus the downstream Cre recombinase can be expressed at the moment; and tissue-specific promoters are added in front of the expression plasmids of Cre and Flp, thus the tissue Cre which is expressed in both the promoters can be expressed. The invention has the beneficial effects that: improvement and innovation are carried out on a traditional building method of a tissue-specific Cre transgenic carrier; and the correct tissue specificity and time specificity of Cre are calibrated by utilizing an intersection of two specific promoters, thus promoting the research on gene function utilizing conditional gene knockout mice and further being applied to research of medical and pharmaceutical targets.

The invention belongs to the technical field of animal gene engineering, and particularly relates to a marking-free porcine beta-alexin 2 gene fixedpoint knock-in plasmid vector and an application thereof. The marking-free porcine beta-alexin 2 gene fixedpoint knock-in plasmid vector disclosed by the invention is relevant to the field of transgenic animals. A donor plasmid pcCAG-R26-PBD2-T2A-PBD2which can express dual-copy pbd-2 genes is constructed, and the donor plasmid further contains a neomycin resistant gene neoR expression box having loxP sequences on two sides. Experiment certificatesthat when the plasmid and a targeting plasmid pX330-pRosa26 which can express Cas9 protein and guide RNA targeting a porcine Rosa26 site at the same time are electrically shifted into recipient cellsat the same time, fixedpoint integration of a pbd-2 gene into the porcine Rosa26 site is realized; through Cre recombinase, resistant marking is deleted; and through somatic nucleus transplantation and embryo transplantation, pbd-2 gene fixedpoint knock-in pigs free from resistant marking can be obtained.

The invention discloses a construction method of recombinant bacillus subtilis of high-yield pullulanase. The method comprises the steps that acid and heat resistance pullulanase gene is optimized by a codon, then is spiced artificially with two different secretory peptides and promoters to form a artificial operon and integrated into Bacillus subtilis A164; integration sites are the positions at which alkaline protease aprE and amylase gene amyE locate respectively; in addition, by the plasmid expression of Cre recombinase , resistance gene integrated into A164 is knocked down; pullulanase fermentation enzyme activity of pullulanase recombinant production bacterial strain CNP2 exceeds 1000 U / mL.

Construction of a recombinant adenovirus vector of an NRIP1 (nuclear receptor interacting protein 1) gene of Qinchuan cattle can be applied to gene function research and dentification on Qinchuan cattle NRIP1, reconstruction of seed cell, regulation on gene metabolism of muscle, fat and reproduction, etc. The Qinchuan cattle NRIP1 gene is inserted under a CMV promoter of a pAdTrack-CMV adenovirusshuttle plasmid to construct a recombinant shuttle plasmid pAdTrack-CMV-NRIP1. After linearization, a PmeI transforms an E.coli BJ5183 competent cell containing a pAdEasy-1 plasmid; an efficient homologous recombinant system of a Cre recombinase in the E.coli BJ5183 is utilized to complete homologous recombination of pAdTrack-CMV-NRIP1 and pAdEasy-1 in bacteria. A correct recombinant adenovirus plasmid is named as pAd-NRIP1. After digestion linearization of pAd-NRIP1 by a PacI, a liposome is transfected to a 293A cell, so as to obtain a recombinant virosome.

The invention relates to the field of biotechnology and discloses a high-proliferation specific recombination eucaryotic cell promoter PCNATAI; an eucaryotic cell operating system PCNATAI-LoxP-stop-LoxP taking a PCNATAI as a core transcription initiator and a LoxP-stop-LoxP as a transcriptional repression element; and a set of general scheme for eliminating the repression effect of the LoxP-stop-LoxP to the transcription process of the PCNATAI promoter through a Cre recombinase provided by a tumor cell specific dependent model trans form and enabling the downstream exogenous tumor suppressor gene or the suicide gene to be effectively and specifically expressed in the malignant tumor cell in a tumor cell specific pattern and a proliferation specific pattern. The scheme not only can remarkably improve the tumor inhibition rate during the gene therapy of the malignant tumor or the expression efficiency of the suicide gene in the malignant tumor cell, but also can ensure the security of the gene therapy.