12 results about "Tyrosine recombinase" patented technology

This invention discloses a novel site-specific recombinase-mediated multisite chromosome editing tool and its applications. The invention provides a complete product suite for integrating or continuously integrating the genome of a host cell, comprising: a circular vector 1 composed of the following elements: loxF1, nox, ori, resistance gene, lox71, P3, P1, P2; and / or a circular vector 2 composed of the following elements: lox66, nox, ori, resistance gene, loxF2, P3, P1, P2; and a circular vector 3 for expressing Nigri and Cre recombinases; and a site-specific knock-in tool for knocking in the lox66-nox fragment at the desired integration site in the host cell genome; P1 and P2 are adapters, used to insert the DNA fragment to be integrated, and P3 is a universal primer. This invention enables rapid and continuous genome integration using simple molecular biology operations.

The present application provides a method for directional transfer of heterologous chromosomes, which comprises the following steps: artificially adding a functional intron containing a loxP sequence into a drug resistance gene, splitting the drug resistance gene into two parts with an overlapping region containing a loxP sequence, inserting the two parts of the drug resistance gene into target chromosomes of a donor species and a recipient species respectively (the two parts of the drug resistance gene undergo Cre / loxP site-specific recombination under the action of a Cre recombinase, and the drug resistance function is recovered), and realizing directional transfer of heterologous chromosomes by means of heterologous cell fusion. The method can be widely applied to gene function research, construction of human disease models, development of therapeutic antibodies, and pharmacological and pharmacodynamic tests.

The invention relates to the technical field of animal models, and particularly discloses a construction method and application of a mouse model with fat dystrophy complicated with atherosclerosis, and the construction method comprises the following steps: mating MED1flox / flox mice with Adipoq-CreERT2 mice to obtain F1-generation mice; the F1-generation mice and MED1flox / flox mice are subjected to backcross, the mice with the genotype being MED1fl / flAdipoq-Cre < + / -> are screened, tamoxifen is injected to induce fat cell specific Cre recombinase expression, and fat cell specific MED1 gene knockout mice are obtained; and performing caudal vein injection of AAV8-PCSK9 virus and feeding with a high-cholesterol and high-fat feed to obtain the fat dystrophy complicated with atherosclerosis mouse model. According to the method provided by the invention, the mouse model with fat dystrophy complicated with atherosclerosis is successfully constructed and obtained, and collaborative simulation and regulation of phenotypes of two diseases are realized.

The application discloses a stringent induction system for corynebacterium glutamicum and application thereof, and belongs to the fields of genetic engineering and microbiology. The system can be strictly regulated without an inducer and can be expressed after the inducer is added. More specifically, it relates to reducing the leakage expression of Cre recombinase by containing logic gates, repressor regulation, and RNA regulatory elements, and its use for mediating the production of large-scale genomic random rearrangement of SCRaMbLE in the synthetic genome of corynebacterium glutamicum. In particular, it is used for reducing the leakage expression level of Cre recombinase and maintaining a moderate expression level after induction. More importantly, the stringent induction system of the application can be applied to obtain rearranged strains with highly diverse genotypic sequences in genomic rearrangement, so as to achieve the purposes of improving the yield of target products or enhancing the stress resistance of strains.

The invention discloses a construction method of a microglial cell conditional knock-down dynactin animal model, which comprises the following steps: selecting a Dctn1LoxP gene knock-in mouse, hybridizing the Dctn1LoxP gene knock-in mouse with a Cx3cr1-Cre transgenic mouse, and carrying out three rounds of breeding to obtain a genotype Dctn1LoxP / LoxP; a mouse of Cx3cr1-Cre; the expression of Cre recombinase is driven by a Cx3cr1 gene promoter, and the 2nd to 4th exons of the Dctn1 gene are cut, so that the knockout of p150Glue and the reduction of the protein levels of other subunits of dynactin are realized. The model is strong in specificity and high in knock-down efficiency, animals show microglial cell morphology abnormity and movement coordination function defects, the model can be used for researching the influence of dynactin on microglial cell neurobiological functions, and a reliable tool is provided for pathogenesis exploration and drug screening of related neurological dysfunction.

The invention relates to the technical field of animal model construction, in particular to a construction method and application of a gene editing mouse model for studying calcium library function and low fertility in mature sperms.The model is characterized in that tamoxifen is used for inducing, a Ddx4ERT2 promoter is used for driving Cre recombinase expression, three subtypes of IP3R are specifically knocked out in sperms at the same time, and the mouse model is obtained. The method is used for researching the action and mechanism of the subtype in male fertility. After the model mouse is subjected to two rounds of induced knockout, the fertility is severely damaged, the sperm quantity and movement level are reduced, and the acrosome reaction is abnormal, so that the model is a good model for evaluating the sperm dysfunction, and is expected to be used for screening treatment drugs for improving the sperm dysfunction, especially male sterility diseases caused by advanced acrosome reaction in the future. In addition, through screening optimization of an IP3R agonist and a calcium signal detection platform, it is further confirmed that the mouse model is a good model for studying sperm calcium library mediated calcium signals.

The invention discloses a construction method and application of a lung cancer bone metastasis non-human animal model. The construction method comprises the following steps: step 1, constructing a Rosa26 fixed-point human-derived SLC7A7 conditional overexpression mouse model; 2, the R26LSL-slc7a7 homozygous mouse and KrasLSL-G12D / + are subjected to a reaction, and the mouse is subjected to a reaction; the method comprises the following steps: carrying out mating on a Tp53flox / flox mouse, carrying out multiple times of selfing on the obtained heterozygote to obtain a KPS mouse, and delivering an effective dose of Cre recombinase to the lung of the mouse. The invention successfully constructs a genetic engineering animal model capable of inducing three-gene collaborative manipulation, the model can efficiently and stably simulate the whole process of lung adenocarcinoma generation, development and distal bone metastasis, and provides a foundation for deeply exploring the pathogenic mechanism of lung cancer bone metastasis, exploring novel diagnostic markers and developing efficient anti-tumor drugs. And a stable and reliable experimental tool with good clinical correlation is provided.

The invention relates to the technical field of biological medicine, and discloses a method for constructing a Myh6-CRE transgenic golden hamster model and application of the Myh6-CRE transgenic golden hamster model. The invention provides a specific DNA (deoxyribonucleic acid) molecule. The specific DNA molecule sequentially comprises an upstream insulator, a Myh6 promoter, a Cre recombinase coding gene, a beta-globin polyA and a downstream insulator from upstream to downstream. The invention also provides a method for preparing the transgenic golden hamster, which comprises the following steps: introducing the expression construct containing the specific DNA molecule into a golden hamster fertilized egg in a fertilized egg microinjection manner, and randomly integrating the expression construct into a golden hamster genome, thereby obtaining the transgenic golden hamster. The invention further provides a Myh6-CRE transgenic golden hamster model, the transgenic golden hamster expresses Cre recombinase under the driving of a Myh6 promoter, and specific recombination between LoxP sites can be mediated. The Myh6-CRE golden hamster constructed by the invention can be used for establishing a conditional gene knockout model, and is suitable for researching the function and action mechanism of the Myh6 related gene in the occurrence and development process of heart and related diseases.

The application discloses a screening marker recovery technology based on a Cre-LoxP combined Flp-Frt recombination system and application. The Cre-Loxp recombination system comprises a Cre recombinase and a LoxP site, and the LoxP nucleic acid combination comprises LoxP71 and LoxP66 sequences which can be specifically recognized and recombined by the Cre recombinase; similarly, the Flp-Frt recombination system comprises a Flp recombinase and a Frt site, and the Frt site comprises a Frt32 sequence which can be specifically recognized and recombined by the Cre recombinase. Due to the lack of screening markers and inducible promoters in the Schizochytrium, the site-specific recombinase cannot be recovered after being integrated into the genome, and therefore, the application aims to combine the two specific recombinase systems, so that the screening markers and the site-specific recombinase can be recovered unlimited times, and thus, the multi-gene editing of the Schizochytrium can be realized.

The invention belongs to the technical field of synthetic biology and genetic engineering, and particularly discloses an anti-leakage Cre recombinase expression cassette and application thereof in pichia pastoris gene editing. The expression cassette is composed of lox71-selection marker-sh blele-Cre coding sequence (SEQ ID NO: 1)-TetO manipulation sequence-lox66, wherein the Cre coding sequence (SEQ ID NO: 1) is optimized by pichia pastoris codon and is introduced into escherichia coli rare codon, and IIS type restriction enzyme sites such as PaqCI, BsmBI, BsaI, AarI and the like are completely eliminated; the gene is cloned to a pPICZ skeleton and introduced into an escherichia coli host of chromosome integrated tetR, Cre leakage expression can be thoroughly inhibited in a large-scale amplification stage, and recombination toxicity and plasmid mutation are avoided. An'upstream homologous arm-expression cassette-downstream homologous arm 'edit box can be obtained through one-step assembly by using Golden Gate, and construction is completed within 3 days; electrically transforming pichia pastoris after linearization, and integrating efficiency gt for the first time; according to the method disclosed by the invention, the content of the resistance marker is 70%, the resistance marker removal rate is 100% through methanol-induced Cre self-deletion, and only one lox71 / 66 heterozygous site is reserved. According to the invention, the problems that the traditional Cre / loxP system is easy to mutate in escherichia coli and low in efficiency in yeast are solved, and an efficient and safe universal gene editing tool is provided for industrial microbial metabolism engineering.

The invention relates to the technical field of neuroscience, in particular to a method for marking a new myelin sheath in a central nervous system, which comprises the following steps: constructing an ENPP6-P2A-CreERT2-T2A-CFP gene segment, and obtaining an ENPP6-CreERT2 knock-in mouse through a gene editing technology; the ENPP6-CreERT2 mouse and the mT / mG report mouse are subjected to mating, and ENPP6-CreERT is obtained; an mT / mG double transgenic mouse; carrying out tamoxifen induction treatment on the double transgenic mice in a target time period, and activating Cre recombinase; brain tissues are collected after induction, and marked middle-stage oligodendroglia cells and myelin sheath structures formed by the middle-stage oligodendroglia cells are observed through a fluorescence report system. According to the invention, the purpose of specifically marking new myelin sheath during tamoxifen induction is achieved. And a new research tool is provided for controlling new myelin sheath in a specific time period. The problems that all new myelin sheaths after induction start can be marked in an existing method, and the new myelin sheaths in the induction period cannot be specifically marked are solved.

The invention provides a construction method and application of a bifluorescence reporter gene animal model for tracking transdifferentiation from airway cilia cells to mucous cells. A transgenic animal model capable of specifically and permanently marking and tracking airway ciliary cell fate transformation is constructed by utilizing a Cre-LoxP gene recombinase system and combining with double promoters and double fluorescence reporter genes. In the progeny, normal cilia cells only express GFP (green). When the cell starts to express MUC5AC under external stimulation (such as PM2.5 and allergen), the existing Cre recombinase in the cell nucleus can recognize and cut off the loxP-Stop-loxP sequence at the downstream of the MUC5AC gene, so that the expression of the RFP gene is started. Finally, mucus-cilia cells obtained by transdifferentiation of cilia cells can simultaneously express GFP (green fluorescent protein) and RFP (yellow), or only express RFP (red) after completely losing cilia cell characteristics. The irreversible color conversion provides a definite evidence for the'provenance and today 'of the cells.