94 results about "U6 promoter" patented technology

The invention discloses a site-specific insertional inactivation method mediated by agrobacterium tumefaciens and CRISPR / Cas9. Endogenous snRNA promoter of Ustilago scitaminea (u6 promoter of Ustilago scitaminea) is used to drive sgRNA expression cassette. CRISPR / Cas9 system integrates with Ti plasmid of agrobacterium tumefaciens to construct the Ustilago scitaminea site-specific insertional inactivation system mediated by agrobacterium tumefaciens with hygromycin as a selection marker. Specific sequence of the objective gene is cloned into sgRNA expression cassette for transformation of Ustilago scitaminea basidiospores, thus the mobile DNA fragment is exactly inserted into the objective gene sequence of Ustilago scitaminea, achieving the goal of damage to gene function. The invention provides an important tool for study of Ustilago scitaminea functional genomics. The system has the advantages of high efficiency and accuracy, and is convenient to use to conduct gene function researches in Ustilago scitaminea.

The invention discloses a DNA fragment with functions of a promoter and application of the DNA fragment. The DNA fragment is any one of the following sequences of (a) a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO.2 or a complementary sequence of the nucleotide sequence; (b) a nucleotide sequence which is obtained by conducting substitution or deletion or addition of one or more nucleotides on the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO.2 and has the functions same as those of the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO.2 as the promoter or a complementary sequences of the nucleotide sequence. The DNA fragment has the functions of the promoter and has the very high specific expression activity, expression of gRNA in a CRISPR-Cas9 system can be achieved on the condition that an inducer does not need to be added, and therefore a U6 promoter from aspergillus niger can be applied in a CRISPR-Cas9 system of the aspergillus niger.

The invention discloses a backbone plasmid carrier for genetic engineering. In the backbone plasmid carrier, a guide RNA (Ribonucleic Acid) expression frame and a Cas9 nuclease expression frame are positioned in one same double-element carrier; the guide RNA expression frame sequentially consists of a rice U6 promoter, a spectinomycin resistance gene, an artificially synthesized sgRNA backbone sequence and a Poly-T terminator; the Cas9 nuclease expression frame sequentially consists of a ZmUBI promoter, a rice preference codon modified Cas9 coded sequence and a 35s terminator. The invention further discloses a recombinant carrier which is established by using the plasmid carrier and contains a target sequence, and an application of the recombinant carrier in rice gene targeting.

The invention discloses a Cas9-mediated bombyx mori gene editing carrier which comprises a constitutive gene editing carrier body and an inducible gene editing carrier body. The constitutive gene editing carrier body comprises an expression cassette U6-sgRNA with a U6 promoter regulating sgRNA expression, and an expression cassette with an IE-1 promoter regulating Cas9 expression. Expression of the inducible gene editing carrier body Cas9 is regulated by an Hr3-39k inducible promoter. A target gene can be stably and efficiently edited through the Cas9-mediated gene editing carrier, and a powerful tool is provided for preparing insect antiviral genetically modified materials and efficient antiviral varieties.

The invention belongs to the technical field of gene engineering, particularly relates to a rubber tree RNA (ribonucleic acid) polymerase type III promoter, in particular to a rubber tree U6 gene promoter proHbU6.6 and further discloses a cloning method and an application of the rubber tree U6 gene promoter proHbU6.6. The rubber tree RNA polymerase type III promoter, namely the rubber tree endogenous U6 promoter proHbU6.6 is obtained by cloning in a Para rubber tree for the first time; the promoter is a rubber tree endogenous RNA polymerase type III promoter, has high transcriptional activity,and can drive expression of downstream sgRNA (small guide ribonucleic acid); the activity of the promoter and feasibility of the promoter applied to a rubber tree CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated nuclease 9) gene editing system are demonstrated by instantaneous conversion of rubber tree protoplast; and CRISPR / Cas9 mediated rubber tree genometarget editing is achieved.

The invention belongs to the technical field of gene engineering, and relates to a rubber tree RNA polymerase III type promoter, in particular to a rubber tree U6 gene promoter proHbU6.8. The invention further discloses a cloning method and application of the promoter. The rubber tree RNA polymerase III type promoter-rubber tree endogenous U6 promoter proHbU6.8 is obtained through cloning in a Brazil rubber tree for the first time. The promoter is the rubber tree endogenous RNA polymerase III-type promoter, the promoter has high-efficiency transcriptional activity, the expression of downstreamsgRNA can be driven, the activity of the promoter and the application feasibility of the promoter in a rubber tree CRISPR / Cas9 gene editing system are verified through transient transformation rubbertree protoplasm, and CRISPR / Cas9-mediated rubber tree genome targeted editing is achieved.

The invention relates to plication-deficient recombinant adenovirus which contains the expression sequence of siGNA of the gene APE1 of human being. The sequence of siGNA of the gene APE1 of human being at which the two ends are provided with restriction site is chemically synthesized and inserted under the prompter U6 of the shuttle plasmid of adenovirus after restriction enzyme, then shuttle plasmid pDC316-EGFP-U6-APE1siRNA is constructed, and then shuttle plasmid pDC316-EGFP-U6-APE1siRNA and the bone plasmid of adenovirus pBHG-fiber5 / 35 are simultaneously cotransfected into cell 293, consequently the recombinant adenovirus Ad5 / F35-APE1siRNA with the expression sequence of siGNA of the gene APE1 of human being is achieved through the fixed-point recombination produced by the system function of Cre / loxP. The recombinant adenovirus is capable of effectively preventing the expression of the gene APE1 and effectively strengthening the sensibility of radiotheraphy and chemotherapy of tumour cell.

The invention discloses a preparation method and an application for a dual-fluorescence reporting system for detecting a micro-RNA (ribonucleic acid) function, wherein the preparation method comprises the following preparation steps of: A, obtaining a mCherry gene sequence via a PCR (polymerase chain reaction) amplification, inserting a rho EGFP-C1 carrier to replace the EGFP (enhanced green fluorescent protein) sequence of the mCherry gene sequence, and constructing a carrier rho mCherry-C1; B, using a RNAi-Ready rho SIREN-RetroQ plasmid as a template, obtaining a human U6 promoter gene via a PCR amplification, and inserting the carrier rho mCherry-C1 to obtain a plasmid rho hU6-mCherry-C1; and C, obtaining a gene sequence carried with a CMV (cytomegalovirus) promoter and an EGFP expression dialog sequence in an interval of 358 to 1944 on a plasmid rho Adtrack-CMV, and inserting the sequence in the plasmid rho hU6-mCherry-C1 to obtain a plasmid rho MGhU6. The plasmid rho MGhU6 is a dual-fluorescence reporting system simultaneously containing a mCherry reporting gene, an internal reference EGFP, a micro-RNA and the target sequence insertion site thereof. The reporting system can be used for detecting the inhibition function of the micro-RNA to the target sequence expression. The detection method is easy, as well as simple and convenient in operation; and a function detection for the micro-RNA can be realized by transfecting one plasmid only. The system also can be used for visually detecting the inhibition function of a micro-RNA in a single living cell to the target thereof by the aid of fluorescence microscopic imaging.

The present invention relates to vectors, compositions and methods for conditional, Cre-lox regulated, RNA interference. Vectors for use in conditional expression of a coding sequence based on a strategy in which the mouse U6 promoter is modified to include a hybrid between a LoxP site and a TATA box, and their use in conditional expression in transgenic mice are disclosed. The vectors allow for spatial and temporal control of miRNA expression in vivo.

The invention belongs to the technical field of gene engineering, particularly relates to an asparagus RNA polymerase III type promoter, more particularly relates to an asparagus U6 gene promoter AspU6P3, and further discloses a cloning method and application of the asparagus U6 gene promoter AspU6P3. The asparagus RNA polymerase III-type promoter, namely the asparagus endogenous U6 promoter AspU6P3, is cloned from asparagus (aka the king of vegetables) cultivated species for the first time; the promoter is the asparagus endogenous RNA polymerase III-type promoter and has efficient transcriptional activity, and can drive expression of downstream sgRNA; in addition, The activity of the endogenous U6 promoter AspU6P3 and the feasibility of the endogenous U6 promoter AspU6P3 applied to an asparagus CRISPR / Cas9 gene editing system are verified through an asparagus stable genetic transformation system, and CRISPR / Cas9 mediated asparagus genome targeted editing is realized for the first time.

The invention provides an adeno-associated virus recombinant vector for knocking out Egr3 gene, and a construction method and application thereof. The vector includes at least the following operably connected sequence elements: a 5' end inverted repetitive sequence, a CMV promoter, a sacas9 coding sequence, a polyA signal sequence, a U6 promoter sequence, a gRNA sequence and a 3' end inverted repetitive sequence. The adeno-associated virus recombinant vector can efficiently knock out Egr3. Through researches, the inventors found that the Egr3 gene knockout vector can effectively inhibit the growth of transplanted tumors and tumor angiogenesis of a HepG2 cell hepatoma model. This shows that the technical scheme of the invention can be applied to the treatment of liver cancer diseases.

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for editing cotton genes. A CRISPR-Cas9 system for cotton genome editing and a construction method thereof are disclosed. According to the method, two upland cotton U6 promoters pGhU6-7 and pGhU6-9 are obtained by cloning, and Bsa I restriction site is mutated. Two vectors pRGEB32-GhU6.7-NPTII and pRGEB32-GhU6.9-NPT II of the CRISPR-Cas9 vector with editing capability in cotton are constructed using an optimized promoter. A cotton endogenous gene GhCLA is selected as a target gene, and four sgRNAs targeting the gene are designed to guide the Cas9 protein to cleave at the specific site of GhCLA. The whitening phenotype, enzymatic map and sequencing of cotton showed that a CRISPR / Cas9 gene editing system driven by pGhU6-7I and pGhU6-9I has editing capability in cotton.

The invention discloses gene medicine for treating hepatitis B and a preparation method thereof. The medicine is composed of a carrier, a U6 promoter and a sequence element expressing Cas9 protein and a sequence element expressing sgRNA, wherein the carrier is used for loading the U6 promoter, a Cas9 sequence and a sgRNA sequence and making the U6 promoter, the Cas9 sequence and the sgRNA sequence be expressed in cells. The Cas9 protein expressed in the gene medicine conducts shearing on cccDNA of HBV under the guidance of sgRNA and further destroys cccDNA, the purpose of being clear about HBV is further achieved, and the problem that a current HBV therapeutic method cannot eradicate HBV is solved.

The invention discloses an aptazyme modified sgRNA carrier with theophylline regulated expression. Aptazyme P1-F5 is inserted to tetraloop and stem loop 2 positions of an expressed sgRNA-AZ 2.0 skeleton; by means of Kpn I and EcoR I sites, and EcoR I and Spe I sites, a U6 promoter and the sgRNA-AZ 2.0 skeleton are connected into the carrier pUC19 / EKSHL in order to obtain the carrier pU6-sgRNA-AZ 2.0; using Bsa I for enzyme digestion of the carrier pU6-sgRNA-AZ 2.0, and using cohesive end design and connecting an sgRNA sequence directing at the target gene so as to obtain the pU6-sgRNA-AZ 2.0-target site. The carrier can effectively mediate the genome editing function of Cas9 protein, and bring the editing activity under regulation of theophylline, also can mediate the transcription inhibition of dCas9-KRAB protein to the target gene, and enables regulation of inhibitory activity by theophylline.

The invention belongs to the technical field of gene engineering, particularly relates to a rubber tree RNA polymerase III-type promoter, more specifically relates to a rubber tree U6 gene promoter proHbU6.1, and further discloses a cloning method and applications thereof. The invention clones and obtains the rubber tree RNA polymerase III-type promoter-rubber tree endogenous U6 promoter proHbU6.1in hevea brasiliensis for the first time, the promoter is rubber tree endogenous RNA polymerase III-type promoter, the promoter has high-efficiency transcription activity and can drive downstream sgRNA expression, the activity and the feasibility of the promoter for application in rubber tree CRISPR / Cas9 gene editing system are verified by transient transformation of rubber tree protoplasts, andCRISPR / Cas9 mediated rubber tree genome targeted editing is realized.

The invention discloses a recombinant baculovirus vector resistant to host apoptosis, which is an antiapoptosis baculovirus expression vector realized by expressing siRNA of an insect cell caspase-1 through the vector. The baculovirus vector contains a section of special DNA sequence, the section of DNA sequence comprises a U6 promoter and an siRNA sequence of caspase-1 targeted by downstream 21nt thereof. A recombinant virus prepared by utilizing the vector can express double-strand small RNA in a host cell, caspase-1 coded by the host cell is silenced in an RNA interference way, and the apoptosis of the host cell can be further inhibited. The recombinant baculovirus vector can be used for constructing a recombinant baculovirus expressing an exogenous gene; after an insect cell is infected with the obtained recombinant baculovirus, the host apoptosis can be inhibited to remarkably enhance the heterologous protein expression level, thereby reducing the production cost.

This invention discloses a method for constructing shRNA interference plasmid targeting human fibrinogen-like protein 2 (hfgl2) and its medical application. The method comprises: screening the target sequence according to hfgl2 cDNA sequence, designing a template of hfgl2 cDNA according to the target sequence, designing a reverse primer containing the template, performing PCR to amplift U6 promoter, inserting BamH I and Hind III sites at both ends respectively, cutting pMSCVneo plasmid with BamH I and Hind III, and inserting U6 promoter sequence with shRNA template sequence at the downstream into pMSCVneo. It is proved by cell level experiments that hfgl2 shRNA interference plasmid can effectively inhibit the expression of hfgl2 gene.

The invention discloses a novel CRISPR Cas13a-gRNA expression vector and application thereof. The novel CRISPR Cas13a-gRNA expression vector is formed by respectively controlling expression of Cas13aand gRNA by virtue of an NF-kB specific promoter DMP and a U6 promoter; a gRNA 5' terminal of the Cas13a-gRNA expression vector is a 28nt sequence capable of targeting mRNA of a specific gene, and generated gRNA is capable of guiding binding of Cas13a proteins and cutting the mRNA of the specific gene, thereby causing expression inhibition of the specific gene. The constructed novel CRISPR Cas13a-gRNA expression vector is capable of specifically knocking down expression of cancer-promoting genes in cancer cells so as to cause growth inhibition and apoptosis of the cancer cells, but expressionand growth of target genes of normal cells are not influenced; and the expression vector can be applied to preparation of novel gene therapy reagents for cancers.

The invention relates to a recombinant plasmid for treating prostatic csarcinoma and melanoma, wherein the GRIM19 expression sequences are located under the CMV promotor of the vector PcDNA3.1(+) and linked by Kpn I and EcoR cleavages, the U6 promotor-siRNA-stat3 specific sequence is located in front of the PcDNA3.1(+)CMV promotor, and linked by Bgl II and NruI cleavages.

The invention provides a double-promoter and double-expression specific shRNA (Short Hairpin Ribonucleic Acid) lentiviral vector and application thereof. The double-promoter and double-expression specific shRNA lentiviral vector comprises an U6 promoter with a nucleotide sequence shown as SEQ ID NO: 1, and an H1 promoter with the nucleotide sequence shown as SEQ ID NO: 2. The experiment results show that the lentiviral vector can express two types of shRNA at the same time, can inhibit the expression of a vpr (Viral Protein Regulatory) gene and / or reduce the expression level of a tat (Transactivator) gene, and can be used for developing and preparing a medicine for preventing HIV (Human Immunodeficiency Virus) infection from occurring.

The invention provides a liver cancer tissue specific RNA (Ribose Nucleic Acid) interference system, as well as a construction method and an application method thereof. The liver cancer tissue specific RNA interference system comprises an AFP (Alpha Fetal Protein)-Cre vector and an LoxP-shRNA vector which are both constructed by lentiviral vectors, wherein the AFP-Cre vector contains an AFP promoter and a Cre recombinase gene placed at the downstream of the AFP promoter; the LoxP-shRNA vector contains a reconstructive H6 promoter and an shRNA sequence placed at the downstream of the U6 promoter; the reconstructive U6 promoter contained in the LoxP-shRNA vector is the U6 promoter in which a structure, capable of indicating whether the AFP-Cre / LoxP-shRNA system is in work, is inserted inside; and the structure, capable for indicating whether the AFP-Cre / LoxP-shRNA system is in work, is shown as LoxP-CMV-eGFP-LoxP. The RNA interference system, provided by the invention, has the characteristics of being high in liver cancer tissue specificity, long-lasting in effect, high in efficiency, excellent in indication performance, and simple and convenient in operation; and the liver cancer tissue specific RNA interference system enables the liver cancer RNA interferential targeted treatment of liver cancer, provides high convenience to relative research and analysis, and can be widely applied to the treatment of the liver cancer and the basic research.

The invention discloses a TNFR1 gene recombinant adenovirus. The adenovirus contains siRNA expressed by an interference TNFR1 gene of a nucleotide sequence shown in SEQ ID No.1, the siRNA is connected to the place below a U6 promoter of shuttle plasmid pHBAd-U6-GFP to prepare an adenovirus shuttle plasmid, and then the adenovirus shuttle plasmid and a skeleton plasmid pHBAd-BHG are subjected to cotransfection 293 cells to obtain adenovirus Ad-TNFR1 shRNA. The TNFR1 gene recombinant adenoviru is used for blocking or reducing TNFR1 expression and blocking a biological effect produced through TNF in a new way, so that tool medicine is designed to explore the effect of TNFR1 in hyperalgesia generation and the intracellular action mechanism, and medicine for preventing inflammatory factors from activating a receptor is designed to weaken the hyperalgesia effect caused after receptor activation.

The invention discloses a biological preparation for resisting against replication and infection of a foot-and-mouth disease virus and a preparation method as well as application thereof. Active parts of the biological preparation are siRNA-VP1 and siRNA-2B; a nucleotide sequence of a sense strand of a transcription template of the siRNA-VP1 is shown as SEQ ID NO. 1; and a nucleotide sequence of a sense strand of a transcription template of the siRNA-2B is shown as SEQ ID NO. 3. In the invention, by connecting the transcription template of the siRNA-VP1 and the transcription template of the siRNA-2B with U6 promoter, human replicated defect 5 type adenovirus plasmid carrier pAdeno-X is cloned to obtain plasmids which can be transcribed and connected in series respectively; and next, cell packaging is performed through packaging to obtain recombinant adenovirus Adeno-2B-VP1. Experiments prove that the prevention and control effect of the recombinant adenovirus Adeno-2B-VP1 is superior to the effect obtained by independently applying the recombinant adenovirus siRNA-2B and the recombinant adenovirus Adeno-2B-VP1 or applying the recombinant adenovirus siRNA-2B and the recombinant adenovirus Adeno-2B-VP1 in a combined way. The biological preparation can be used for suppressing replication of the foot-and-mouth disease virus and resisting against the infection of the foot-and-mouth disease.

The invention relates to a usage of a replication-defective recombinant adenovirus which contains the expression sequence of human APE1 gene siRNA, and the invention can be used in the preparation of the drugs to play sensitizing effect in the radiotherapy and / or chemotherapy of malignant tumors. The expression sequence of human APE1 gene siRNA is inserted below the U6 promoter of adenovirus shuttle plasmid, so as to construct shuttle plasmid pDC316-EGFP-U6-APE1siRNA, and the latter and adenovirus backbone plasmid pBHG-fiber5 / 35 transfect 293 cells in total, and the Ad5 / F35-APE1siRNA recombinant adenovirus containing the expression sequence of human APE1 gene siRNA is obtained by fixed-point recombinant by a Cre / loxP system. The recombinant adenovirus can be made into the injection liquid with certain titer, be used in the combined application in the radiotherapy and / or chemotherapy of malignant tumors, effectively control the expression of APE1 gene and effectively enhance the sensitivity to the radiotherapy and / or chemotherapy of malignant tumors.

The invention discloses a preparation method and nucleic acid construct of male sterile lepidoptera insects. The method comprises the steps that 1, the Ser2 gene knockout nucleic acid construct is built, wherein the nucleic acid construct comprises the following operable connection elements from the 5' terminal to the 3'terminal, and the operable connection elements include one U6 promoter, a first Ser2 genetic target, polyA, another U6 promoter, a second Ser2 genetic target and polyA; 2, the construct and a PHA3PIG plasmid capable of expressing Piggybac transposase are used for co-transformation of fresh lepidoptera insect eggs, a generation G0 is obtained and subjected to selfing, and a generation G1 is obtained; 3, the generation G1 and transgenic lepidoptera insects for expressing Cas9protein are mated to obtain a generation G2. According to the method, a CRISPR / Cas9 technology based on a PiggyBac transposon is used for successfully establishing the male sterile lepidoptera insects on the premise that normal mating behaviors are not affected, and the method has important value in prevention and treatment of lepidoptera pests through the male sterility technology.

The invention discloses a lentiviral vector, wherein the original hUbc promoter in FUGW is replaced by a U6 promoter or a strong promoter EF1alpha, the original EGFP gene is replaced by a strong fluorescent protein copGFP or a Tomato gene, a eukaryon resistance label gene, multiple clone sites and tag protein Flag are linked to the same cistron of the gene, and splicing protein T2A and P2A are adopted to link a plurality of genes on the same cistron. Compared with the conventional lentivirus vector, the lentiviral vector of the present invention has the following advantages that: a plurality of proteins can be concurrently and efficiently expressed with the same cistron, fluorescence intensity is high, induced function recovery is performed on the interfered gene, the vector is suitable for carrying large fragment genes, and the like. In addition, the lentivirus vector has characteristics of wide application, high efficiency, benefits for applications of the lentiviral vector in clinical and science researches, good practical value and good application prospects.

The invention provides a gene editing system based on a CRISPR-Cas9 technology and application of the gene editing system. Based on an endogenous U6 promoter dependent-form vector system, a negative selection marker is added into a Cas9 expression box of an expression vector, an anti-drug selection marker on a rescue vector containing a homologous arm is transferred onto the expression vector, thus the base size of the rescue vector is decreased, and the capacity of the rescue vector is increased; and an exogenous gene segment lengthening out to 6.3 kb can be input in human plasmodium in a mediated mode, obtained recombinant plasmodium does not contain the anti-drug selection marker and Cas9 protein expression plasmid residue, continuous gene editing can be conducted through the same drugselection marker, and the gene editing system is stable in performance, efficient, concise, and powerful in function, and has wide application prospects and huge market value.

The invention discloses an exosome delivery CasRx gene silencing AAV vector. The AAV vector contains CasRx protein expression, and a U6 promoter drives sgRNA expression. The invention discloses a construction method of the exosome delivery CasRx gene silencing AAV vector. According to the construction method, when the AAV vector is separated from a conditioned medium for producing cells, AAV can be combined with exosome exoAAV, and efficient delivery of the gene vector can be realized by wrapping the AAV vector with the exosome. Compared with the prior art, the AAV vector has the beneficial effects that 1, the gene knockout efficiency is higher (greater than 90%); 2, compared with RNA interference (shRNA), the off-target effect is eliminated; 3, the AAV vector has the capability of targeting nuclear RNA (including non-coding RNA); 4, splicing is manipulated, wherein misconnection defects and targeted exon jumping are corrected; and 5, the size is small, a single vector AAV-CasRx can be realized, and RNA can be guided in vivo.

The present invention relates to a novel endogenous gene expression substituting lentivirus vector. According to the present invention, a U6 promoter is inserted between the 5' LTR and the 3' LTR of lentivirus so as to perform transcription of a segment of a shRNA sequence, and the shRNA insertion region contains double restriction enzyme cutting sites such as BamHI and XhoI so as to insert the shRNA sequence after annealing; an EFla promoter is used for transcriptional expression of a Flag tag fused exogenous gene, the Flag tag is fused on the N terminal of the exogenous gene, and the multiple cloning site contains three restriction enzyme cutting sites such as HpaI, EcoRI and NheI3 so as to insert the exogenous gene, wherein the HpaI is the blunt-ended restriction enzyme cutting site so as to ensure insertion of any genes; IRES is used for starting the EGFP reporter gene; and the production of two proteins through the one RNA transcripted from the EFla can be achieved. In addition, the disadvantages of the conventional vectors are overcome, and the fast and efficient intracellular genetically modified way is provided.