823 results about "Plasmid Vector" patented technology

Methods for efficient production of recombinant AAV employ a host cell which comprising AAV rep and cap genes stably integrated within the cell's chromosomes, wherein the AAV rep and cap genes are each operatively linked to regulatory sequences capable of directing the expression of the rep and cap gene products upon infection of the cell with a helper virus, a helper gene, and a helper gene product. A method for producing recombinant adeno-associated virus (rAAV) involves infecting such a host cell with a helper virus, gene or gene product and infecting the infected host cell with a recombinant hybrid virus or plasmid vector containing adenovirus cis-elements necessary for replication and virion encapsidation, AAV sequences comprising the 5′ and 3′ ITRs of an AAV, and a selected gene operatively linked to regulatory sequences directing its expression, which is flanked by the above-mentioned AAV sequences.

Provided herein are methods for generating human induced pluripotent stem cells free from genomic integration of exogenous transgenes by transfecting into nucleated blood cells one or more DNA expression vectors (e.g., plasmid vectors) that do not contain a mammalian origin of replication, and encode and permit expression of one or more reprogramming factors (e.g., Oct4, Sox2, Klf4, and c-Myc). Also provided herein are the integration-free human induced pluripotent stem cells obtained by the methods described herein.

Aerosol delivery of nucleic acids to the lungs using viral vectors, polymers, surfactants, or excipients has been described. Compositions for intranasal administration are described that contain nucleic acids without viral or plasmid vectors and with little to no polymers, surfactants, or excipients. In one embodiment, the composition for intranasal delivery consists essentially of at least one nucleic acid and an aqueous solution. Suitable nucleic acids for intranasal delivery include, but are not limited to, dsDNA, dsRNA, ssDNA, ssRNA, short interfering RNA, micro-RNA, and antisense RNA. Methods for treatment, diagnosis, or prevention of at least one symptom or manifestation of a lung disease are also described consisting of administration by intranasal delivery an effective amount of a composition containing a nucleic acid. The composition may be formulated as a liquid or aerosol or other acceptable formulation for intranasal administration.

The invention mainly belongs to the technical field of gene editing and particularly relates to an SSA (single-strand annealing) repair-based gene seamless editing method utilizing a CRISPR/Cas9 technology. According to the gene seamless editing method, CRISPR/Cas9 expression vectors CCR5.CRISPR/Cas9 and eGFP.CRISPR/Cas9 targeting CCR5 and eGFP are constructed firstly, a donor CCR5-Donor vector is constructed, a CCR5-Donor plasmid vector adopts the pXL-BACII-L-arm-CAG-TK-PGK-Puro<R>-T2A-eGFP-bGH pA-R-arm structure, L-arm and R-arm are left and right homologous arms, and CAG-TK-PGK-Puro<R>-T2A-eGFP-bGH pA between L-arm and R-arm is a screening marking component; through two times of transfection, seamless editing is completed by using HR and SSA repair mechanisms in the CRISPR-Cas9 system and cells through positive and negative screening of puromycin and ganciclovir of the cells.

The invention discloses a miR-205 gene knockout kit based on CRISPR-Cas9 gene knockout technology. According to the invention, an optimal CRISPR-Cas9 target sequence of a certain amount of miR-205 is obtained through target design software and an sgRNA single chain is synthesized in vitro; an insertion fragment is obtained through processing; then sgRNA is inoculated into a plasmid vector by using T4 ligase; and a miR-205 gene knockout cell strain is obtained through transfection of an LNCap cell strain, continuous drug screening and fluorescence detection. Heterogenous hybridization double strands are obtained by extracting DNAs of a cell, PCR amplification of miR-205, purification, denaturation of a PCR product and annealing; T7E1 enzyme digestion test is employed to determining shearing efficiency of a CRISPR-Cas9 system on miR-205; a verified optimized a miR-205 gene knockout CRISPR-Cas9 target sequence is obtained; and the kit is constructed on the basis of the target sequence and can be used for directional knockout of miR-205 genes. The kit has the characteristics of high gene knockout efficiency, fast speed, easiness and economic performance and has wide prospects in the aspects of construction of animal models and clinical research of medical science.

The invention provides a specific sgRNA combined with an immunogene to inhibit HBV replication, an expression vector thereof, and an application of the specific sgRNA and the expression vector. The sgRNA sequences of a human hepatitis B virogene suitable for CRISPR-Cas9 targeting editing and a PD-1 gene are designed, the plasmid vector of the sgRNA inhibiting HBV and PD-1 genes is constructed, and the plasmid vector and a nuclease gene expression vector are transferred to HBV transgenic mice, and can obviously inhibit HBV DNA replication. The gene expression vector prepared in the invention has the advantages of simple method steps, good sgRNA targeting property, and high CRISPR-Cas9 knockout efficiency. The sgRNAs specifically targeting the HBV and PD-1 genes can accurately splice the HBV and PD-1 genes, inhibit in vivo hepatitis B virus replication, and reduce the hepatitis B virus antigen expression.

The invention provides an sgRNA combined with immunogene to inhibit high risk HPV expression, a gene knockout vector thereof, and an application of the sgRNA and the vector. The sgRNA sequences of a human papilloma virus 16 type gene suitable for CRISPR-Cas9 targeting editing and a human PD-1 gene are selected, and the plasmid vector of the sgRNA and a CRISPR-Cas9 nuclease gene expression vector are transferred to HPV16+ human cervical carcinoma cells and HPV16+ transplantation tumor-bearing mice, and can obviously inhibit HPV16 expression and inhibit tumor growth. The gene expression vector prepared in the invention has the advantages of simple method steps, good sgRNA targeting property, high CRISPR-Cas9 knockout efficiency, accurate targeting splicing of high risk HPV16 type and PD-1 genes, efficient reduction of the expression of the high risk HPV16 type gene, and obvious inhibition of the tumor growth through combined application.

A vaccine comprising an immunologically effective amount of recombinant modified vaccinia Ankara (rMVA) virus which is genetically stable after serial passage and produced by a) constructing a transfer plasmid vector comprising a modified H5 (mH5) promoter operably linked to a DNA sequence encoding a heterologous foreign protein antigen, wherein the expression of said DNA sequence is under the control of the mH5 promoter; b) generating rMVA virus by transfecting one or more plasmid vectors obtained from step a) into wild type MVA virus; c) identifying rMVA virus expressing one or more heterologous foreign protein antigens using one or more selection methods for serial passage; d) conducting serial passage; e) expanding an rMVA virus strain identified by step d); and f) purifying the rMVA viruses from step e) to form the vaccine. One embodiment is directed to a fusion cytomegalovirus (CMV) protein antigen comprising a nucleotide sequence encoding two or more antigenic portions of Immediate-Early Gene-1 or Immediate-Early Gene-2 (IEfusion), wherein the antigenic portions elicit an immune response when expressed by a vaccine.

The invention relates to a method for acquiring a Nogo-B knockout mode mouse based on CRISPR/Cas9 technology and an application thereof, the method for acquiring the Nogo-B knockout mode mouse comprises the following steps of: designing a high-efficiency sgRNA for identifying EGE-ZXH-007 of a specific gene; connecting the sgRNA to a plasmid vector and then carrying out in-vitro transcription to obtain a Ca9/sgRNA capable of being subject to microinjection; injecting the Ca9/sgRNA capable of being subject to microinjection into a mouse fertilized egg, and then carrying out genotype detection and identification on the born mouse, and finally obtaining the Nogo-B knock-out mode mouse. According to the invention, the Nogo-B knockout mode mouse is obtained by using the CRISPR/Case9 technology,and compared with the traditional gene knockout technology, the operation is easy and the efficiency is higher, and the homozygous mutation is more easily obtained, thus laying a good foundation for further researching the function of Nogo-B.

The invention discloses a precision and high-efficiency transgenic carrier as well as a construction method and application thereof. The transgenic carrier is obtained by completely assembling a genecoding CRISPR/Cas9 proteins, a DNA template of gRNAs, a homologous arm of a target gene side wing and a target gene to be transferred into a same plasmid vector. By adopting the precision and high-efficiency transgenic carrier as well as the construction method and application thereof, the workload and the subsequent hybridization for operating the gene in a laboratory can be significantly reduced, the homozygote generation time can be shortened, and the transgenic efficiency can be significantly increased. Based on an MCR method, the external target gene and the Cas9 gene are jointly transferred into silkworm genome in the process for precisely modifying the gene by virtue of the catalytic effect of the Cas9, the homozygote can be generated in a G0 generation, i.e. the mutation generatedon one copy can be automatically propagated onto an allele.

The present invention concerns methods and compositions for the construction of a series of stable vectors for genomic library construction useful in Gram negative species. In certain embodiments, the vectors contain the pBBR1 replicon, capable of to stable replication in a broad range of Gram negative species. In various embodiments, the plasmid vectors may also contain bidirectional, rho-independent transcriptional terminators flanking the multiple cloning site, which allows for greater insert stability, and thus, greater genomic representation. Each vector may vary in its selection marker region, mobilization function, and promoter used to express insert sequences. These vectors are of use in the screening of highly representational genomic libraries in a broad variety of Gram negative species.

The invention discloses an LMNA gene knocked-out cell line. The cell line is a 293T cell line or a HePG2 tumor cell line. Cells to be knocked out are transfected by plasmid vectors constructed by twopairs of gRNA as shown in SEQ ID NO. 1-4 or two pairs of gRNA as shown in SEQ ID NO. 7-10 on the basis of a crispr-cas9 technology, and then are subjected to resistance screening to obtain the cell line. The LMNA gene knocked-out cell line can be used for intervention drug screening cell models for diseases such as dilated cardiomyopathy, fat metabolic disorder syndrome and premature aging syndromes.

The invention discloses a stable knockout single plasmid vector with coordination of transposon and a CRISPR/Cas9 system and application of stable knockout single plasmid vector and belongs to the field of gene engineering. The single plasmid vector is a double-stranded circular plasmid containing an IRDR-L-IRDR-R box, and the IRDR-L-IRDR-R box comprises an IRDR-L sequence, a promoter, a gRNA scaffold sequence, a Cas9 protein sequence, a resistance screening gene sequence and an IRDR-R sequence. The single plasmid vector provided by the invention can realize expression of sgRNA and a Cas9 protein only needing once construction and once transfection. A method is simple in process, is efficient and rapid, greatly simplifies a plasmid construction flow, shortens an experiment period and improves the working efficiency. The vector provided by the invention carries a transposase recognition sequence, does not use a virus, can conveniently, rapidly and safely establish a gene knockout stablecell line and is beneficial to screening stable cell lines by comprising the puromycin screening resistance.

The invention relates to a stable recombinant expression plasmid vector comprising a fragment of polynucleotide encoding a recombinant antitoxin gene. The polynucleotide expresses a polypeptide used for neutralizing toxic polypeptide of a host cell. The toxic polypeptide is expressed in a host cell by a fragment of polynucleotide encoding a toxin gene. The recombinant expression plasmid vector also comprises a fragment of polynucleotide encoding polypeptide expression product. The stable recombinant expression plasmid vector is derived by replicable frame plasmid in Hafnia alvei. The invention also discloses a transformant of the stable recombinant expression plasmid, a method for preparing bio-based pentanediamine by using the transformant, and bio-based pentanediamine prepared with the method provided by the invention. The invention also discloses polyamide and a composition containing polyamide, which are prepared by using the bio-based pentanediamine prepared with the method as a raw material. The invention also discloses a method for preparing pentamethylene-1,5-diisocyanate. The method comprises the steps that: bio-based pentanediamine is prepared by using the method provided by the invention; and the bio-based pentanediamine is transformed into pentamethylene-1,5-diisocyanate.

The present invention provides compositions and method for regulating cellular growth and metabolism, intra- and extracellular enzymatic activities, and synthesis of endogenous and/or heterologous proteins, comprising the steps of cloning genes encoding an mRNA interferase (toxin) and its cognate antitoxin; expressing these proteins in a host cell from two separate constitutive or inducible promoters on one or more plasmid vectors or on a chromosome; and regulating the cellular growth and metabolism by controlling the ratio of toxin and antitoxin present in the host cell. Optionally, the method provides further steps of modifying an endogenous or heterologous gene of interest to substitute all mRNA recognition sequences with sequences that are not cleavable by the mRNA interferase being expressed without any change in the amino acid sequence of the protein encoded by the gene; and co-expressing the gene of interest in the same host cell.

The invention relates to the field of biotechnologies, and provides a construction method for a sialidase gene knockout mouse model and application thereof. An in vitro and vivo targeted mouse genomeNPL is used for genetically recombining a CRISPR knockout plasmid vector, and a diploid double-knockout NPL mouse knockout model is successfully constructed through an oosperm microinjection technology. The NPL has the important function in a pathophysiological process of a body. Vicious transformation of a cancer cell is always along with overexpression of sialic acid. Now, the overexpression ofthe sialic acid has become one of important markers of the malignant transformation and transferring of a tumor. The model is capable of constructing a new research platform for researching a processof cell canceration, beneficial to a key step of researching generation and transferring of the tumor, and providing a foundation for the research and development of a novel anti-tumor drug.

According to the invention, through amplifying a strong promoter in Ensifer adhaerens and carrying out bioinformatics analysis and functional verification, a strong promoter is obtained, and the strong promoter can be widely used for gene expression, genetic manipulation and strain improvement in Sinorhizobium meliloti, Zymomonas mobilis, Caulobacter crescentus, Pseudomonas denitrificans, Agrobacterium tumefaciens, Brucella abortus, Pseudomonas fluorescens, Rhizobium leguminosarum, Ensifer adhaerens and other alpha-proteobacteria and has a nucleotide sequence of SEQ ID NO: 1. The invention also relates to a plasmid vector containing the strong promoter, a method for constructing a genetic engineering strain by using the strong promoter, and an application of the corresponding strain and ahost cell in starting expression of a target gene.

The invention relates to a pig breeding and breathing complex virus (PPRSV) and pig 2-type loop virus (PCV 2) series gene recombination adenovirus and vaccine in the field of high and new biotechnology. It uses PCR technology to clone the Cap protein gene into the plasmid carrier pShuttle-CMV-GP5 by open type reading rule and transforms tobacillus coli BJ5183strain with cage carrier pAdEasy-1 to capture the recombination plasmid; it uses recombination plasmid HEK293-A cell to capture recombination adenovirus and purifies it to express the recombination adenovirus rAd-Cap-GP5 of PRRSV GP5protein and PCV-2 Cap protein.

The present invention relates to method for obtaining optimum expressed proteins in a transformed gram positive bacteria by specific integration of T7 RNA polymerase gene into the chromosome of a gram positive bacteria exhibiting resistance to aminoglycosides, said method comprising:- digesting an E. coli plasmid with a restriction enzyme- digesting the genomic DNA of said gram positive bacteria- ligating the said digested plasmid to the digested genomic DNA of said gram positive bacteria- transforming the said gram positive bacteria protoplasts with the said ligation mixture of step 2 to yield transformed gram positive bacteria (transformants),- screening the said transformants for kanamycin resistance and aminoglycoside sensitivity to ensure that the targetting of the said plasmid vector into the chromosome of the said gram positive bacteria is successful- cloning of the desired gene in the said vector—culturing the transformant in a suitable culture medium- isolating the expressed proteins from the culture medium

The invention discloses a tobacco PDS gene fragment-containing cucumber mosaic virus RNA2 attenuated mutant type plasmid vector and application thereof. The plasmid vector contains a CMVFny strain RNA2 complete sequence, and a tobacco PDS gene 199bp fragment is inserted behind a 2a protein termination codon. The plasmid vector and a wild CMVFny RNA1 and wild CMVFny RNA3-containing plasmid are inoculated to nicotiana benthamiana through an agrobacterium infiltration method, so that weakly pathogenic symptoms can be displayed, and the capacity of inducing gene silencing can be judged with nakedeyes without depending on an instrument; great convenience is provided for deep researches on attenuated vaccines.

The invention discloses a gateway cloning technology entry plasmid vector. The vector contains a photoinducible promoter (Prbcs) of a small subunit gene of 1,5-bisphosphate carboxylase (Rubisco) and a green fluorescent protein (GFP) reporter gene. The invention also discloses a construction method and application of the vector. Through the vector, the gateway cloning technology can be utilized to quickly construct a photoinducible plant expression vector of a target gene to realize the high level expression of the target gene in plant leaves, the expression of the target gene is adjusted by light intensity, and expressed target protein can be positioned into chloroplast or cytoplasm.