143 results about "Internal ribosome entry site" patented technology

A method of mRNA production for use in transfection is provided, that involves in vitro transcription of PCR generated templates with specially designed primers, followed by polyA addition, to produce a construct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ cap and/or Internal Ribosome Entry Site (IRES), the gene to be expressed, and a polyA tail, typically 50-2000 bases in length. This RNA can efficiently transfect different kinds of cells. This approach results in increased efficiency (fidelity and productivity) of mRNA synthesis and is less time consuming because it does not require cloning, and also consequently eliminates the unwanted errors and effects related to RNA made on DNA templates obtained with cloning techniques. The results of transfection of RNAs demonstrate that RNA transfection can be very effective in cells that are exceedingly difficult to transfect efficiently with DNA constructs. Further, the levels of gene expression following mRNA transfection are consistent from cell to cell in an experiment and these levels can be controlled over a wide range simply by changing the amount of mRNA that is transfected, and without obvious cytotoxic effects due to the levels of RNA per se. Due to high efficiency the cells can be simultaneously transfected with multiple genetic constructs. The method can be used to deliver genes into cells not- or only poorly transfectable for DNA, in vitro and in vivo.

The present invention provides for transposon vectors encoding expression control region-traps and gene-traps. Also provided are dicistronic vectors. Certain embodiments of the invention contain internal ribosome entry sites.

Synthetic and isolated translational regulatory elements, including oligonucleotides that have translational enhancing activity, internal ribosome entry site (IRES) activity, or translational inhibitory activity, and multimers of such translational regulatory elements are provided. In addition, compositions that include such translational regulatory elements are provided, as are methods of using the translational regulatory elements.

The invention provides a DNA molecule comprising a multicistronic transcription unit coding for i) a polypeptide of interest, and for ii) a selectable marker polypeptide functional in a eukaryotic host cell, wherein the polypeptide of interest has a translation initiation sequence separate from that of the selectable marker polypeptide, and wherein the coding sequence for the polypeptide of interest is upstream from the coding sequence for the selectable marker polypeptide in said multicistronic transcription unit, and wherein an internal ribosome entry site (IRES) is present downstream from the coding sequence for the polypeptide of interest and upstream from the coding sequence for the selectable marker polypeptide, and wherein the nucleic acid sequence coding for the selectable marker polypeptide in the coding strand comprises a GTG or a TTG startcodon. The invention also provides methods for obtaining host cells expressing a polypeptide of interest, said host cells comprising the DNA molecules of the invention. The invention further provides the production of polypeptides of interest, comprising culturing host cells comprising the DNA molecules according to the invention.

A method of creating a nucleic acid sequence having an adenine-rich nucleic acid block of at least 25 nucleotides in length is provided, whereby said nucleic acid sequence is capable of causing translation by internal ribosome entry (IRES element). Said adenine-rich nucleic acid block comprises from 40 to 100 mol-% adenine.

The invention relates to an infectious arenavirus particle that is engineered to contain a genome with the ability to amplify and express its genetic information in infected cells but unable to produce further infectious progeny particles in normal, not genetically engineered cells. One or more of the four arenavirus open reading frames glycoprotein (GP), nucleoprotein (NP), matrix protein Z and RNA-dependent RNA polymerase L are removed or mutated to prevent replication in normal cells but still allowing gene expression in arenavirus vector-infected cells, and foreign genes coding for an antigen or other protein of interest or nucleic acids modulating host gene expression are expressed under control of the arenavirus promoters, internal ribosome entry sites or under control of regulatory elements that can be read by the viral RNA-dependent RNA polymerase, cellular RNA polymerase I, RNA polymerase II or RNA polymerase III. The modified arenaviruses are useful as vaccines and therapeutic agents for a variety of diseases.

Methods and constructs for genetic manipulation of one or more of shrimp, shellfish, mollusks, and fish are disclosed. The nucleic acid construct includes a promoter and an internal ribosome entry site of an insect picomavirus, such as a cricket paralysis-like picomavirus. One or more open reading frames can be operably associated with one or both of the promoter and the internal ribosome entry site, and one or more proteins or protein subunits can be expressed upon introduction of the construct into a host cell, such as into a shrimp. Method for producing immortalized crustacean cell lines using enhancer elements derived from shrimp and/or shrimp viruses are also described.

The invention discloses a late promoter targeting adjustment and control oncolytic adenovirus pCN305 vector, a construction method and application thereof. The vector is established on a pCN103 vector with targeting property; all exogenous genes for killing and inhibiting various cancers, such as IL-24, TRAIL, SOCS3, cpp-SOCS3, SOCS1, cpp-SOCS1and so on, are adjusted, controlled and expressed by late promoters of viruses through induction of internal ribosome into IRES sites and polyclonal sites; and the pCN305 vector has good anticancer effect through experiment in vivo and vitro. All double-target replicating oncolytic adenovirus established by application of the pCN305 vector, such as AdCN305-IL-24, AdCN305-TRAIL, AdCN305-SOCS3, AdCN305-cpp-SOCS3, AdCN305-SOCS1, AdCN305-cpp-SOCS1 and so on, can be used for treating various tumors. Moreover, the invention can develop a novel virus-gene anticancer medicine for effectively treating the tumors.

The invention discloses a recombinant expression vector for stably and efficiently expressing a human beta-NGF (Nerve Growth Factor), a recombinant cell strain containing the recombinant expression vector and a method for producing a recombinant human beta-NGF. The recombinant expression vector comprises a promoter, a beta globin gene intron and a human beta-NGF gene, an internal ribosome entry site sequence and a selective marker gene and has higher integration and expression efficiency in a host cell. A recombinant eukaryotic cell containing the recombinant expression vector expresses the recombinant human beta-NGF equivalent with a natural beta-NGF in activity and still can stably express after multiple times of passage; and the expressed beta-NGF is secreted into a culture medium and is easy to separate and purify, the production downstream difficulty and the production downstream cost are greatly reduced, and the clinical application prospect and the commercial value are good.

A method for conditionally knocking out and altering gene function and genetic sequences that can be used in such methods, for use in gene trapping and gene targeting. Specifically, the genetic sequence is a inducible gene silencer comprising: (a) a splice acceptor sequence; (b) an internal ribosomal entry site (IRES) sequence; (c) a nucleotide sequence coding for a reporter protein; (d) a polyadenylation sequence; and (e) a pair of oppositely oriented recombination site sequences, which cause single cycle inversions in the presence of a suitable recombinase enzyme, flanking elements (a) through (d).

The invention provides a DNA molecule comprising a multicistronic transcription unit coding for i) a polypeptide of interest, and for ii) a selectable marker polypeptide functional in a eukaryotic host cell, wherein the polypeptide of interest has a translation initiation sequence separate from that of the selectable marker polypeptide, and wherein the coding sequence for the polypeptide of interest is upstream from the coding sequence for the selectable marker polypeptide in said multicistronic transcription unit, and wherein an internal ribosome entry site (IRES) is present downstream from the coding sequence for the polypeptide of interest and upstream from the coding sequence for the selectable marker polypeptide, and wherein the nucleic acid sequence coding for the selectable marker polypeptide in the coding strand comprises a GTG or a TTG startcodon. The invention also provides methods for obtaining host cells expressing a polypeptide of interest, said host cells comprising the DNA molecules of the invention. The invention further provides the production of polypeptides of interest, comprising culturing host cells comprising the DNA molecules according to the invention.

The invention establishes a novel expression carrier containing a weakened internal ribosome entry site (IRES for short) sequence and a double-sieving mark by a molecular biology technique. The carrier can be used for the recombinant expression of a human coagulation factor 8, and can effectively enhance the expression quantity of the coagulation factor 8 in the cells of mammals by the comprehensive sieving of a stable expression strain.

The present invention provides a cyclic RNA preferable for carrying out rotary protein translation in which translation domains other than that of the target protein are sufficiently short and translation efficiency is high, and a method for producing protein that uses this cyclic RNA as template. More specifically, the present invention provides a cyclic RNA that encodes a protein, has a full-length number of bases that is equal to or greater than 102 and is a multiple of 3, has at least one start codon, does not have a stop codon in the same reading frame as the start codon, and does not contain an internal ribosome entry site (IRES). In addition, the present invention provides a method for producing protein in a eukaryotic cell expression system that consists of using the aforementioned cyclic RNA as template and expressing a protein encoded by that cyclic RNA.

The invention provides a DNA molecule comprising a multicistronic transcription unit coding for i) a polypeptide of interest, and for ii) a selectable marker polypeptide functional in a eukaryotic host cell, wherein the polypeptide of interest has a translation initiation sequence separate from that of the selectable marker polypeptide, and wherein the coding sequence for the polypeptide of interest is upstream from the coding sequence for the selectable marker polypeptide in the multicistronic transcription unit, and wherein an internal ribosome entry site is present downstream from the coding sequence for the polypeptide of interest and upstream from the coding sequence for the selectable marker polypeptide, and wherein the nucleic acid sequence coding for the selectable marker polypeptide in the coding strand comprises a GTG or a TTG start codon. Also provided are methods for obtaining host cells expressing a polypeptide of interest, such host cells comprising DNA molecules of the invention. Further provided is the production of polypeptides of interest, comprising culturing host cells comprising the DNA molecules of the invention.

The invention relates to compositions, specifically novel nucleic acid constructs encoding a cardiovirus 2A polypeptide operably linked to suitable promoters. Also, disclosed are methods whereby the nucleic acid constructs are introduced into cells or cell free systems to regulate cellular mRNA transcription and cap-dependent or internal ribosomal entry site (IRES)-dependent mRNA translation.

The present invention discloses a nucleic acid construct. The nucleic acid construct is characterized in that the nucleic acid construct has a structure as shown in the formula car-[(IRES)-f]<m>, wherein the IRES is a sequence of an internal ribosome entry site, f encodes a functional protein F, m is 0 or a non-0 natural number, car encodes a CAR, the CAR comprises (a) an extracellular binding domain that specifically recognizes CLDN18.2, (b) a hinge domain, (c) a transmembrane domain, (d) a costimulating intracellular domain, and (e) a signal transduction domain, the extracellular binding domain comprises scFv, and the amino acid sequences and functions of the extracellular binding structural domain are described in the specification. The CAR molecule provided by the invention has excellent safety and effectiveness and shows a very good tumor inhibition effect.

The present invention provides a method and components thereof of performing genetic modification under a drug-free environment. The method comprises the steps of generating a trapped mammalian cell library by trapper constructs (including the element of piggyBac terminal inverted repeats (TIRs)), reporter constructs, and helper constructs (including a sequence of an internal ribosomal entry site (IRES)). The present art allows: (1) to target & identify the silenced loci; (2) to separate genes with low-level expression at certain differentiation stages; (3) to evaluate the efficiency of gene targeting in the silent or repressed loci. The present invention avoids the biased gene targeting observed in the prior arts, and eliminates the needs of introducing antibiotic genes into the host genome which may lead to a potential threat of drifting antibiotic resistant genes into environment.

The invention discloses a recombinant vector for gene knock-in. The recombinant vector comprises a DNA (deoxyribonucleic acid) fragment structure IL17A-IRES-luc, wherein IL17A is an encoding gene of interleukin 17A, IRES is internal ribosome entry site, and luc is an encoding gene of secreting luciferase. The invention also provides a method for preparing the gene knock-in recombinant vector and a mouse model prepared by using the recombinant vector, wherein the mouse model can be used for detecting the expression of the IL17A gene by detecting the expression of the secreting luciferase in blood or urine; a fluorescent microscope or other complicated equipment is not needed; the secreting luciferase emits light by virtue of an oxidizing reaction of substrate coelenterazine in the catalysis of luciferase, and laser light is not needed, so that the fluorescent background is far lower than GFP (green fluorescent protein) fluorescence, the detection sensitivity is higher, the background is cleaner, and the experiment results are more accurate.

The invention relates to a bicistronic mRNA coexpression gene transporter and a preparation method thereof. The gene order of the gene transporter is SEQ ID No. 15; the gene transporter from 5' end to 3' end comprises bovine Nuclear matrix binding region MAR, artificially constructed combined promoter CAG, Profilin gene FSTN, internal ribosome entry site (IRES), green fluorescent protein AcGFP gene and Rabbit globin poly A signal region; the preparation method comprises the following steps: constructing a carrier pCAG-IRES2-AcGFP1; obtaining FSTN gene and inserting the pCAG-IRES2-AcGFP1 carrier; obtaining the sequence of MAR and inserting the pCAG-IRES2-AcGFP1 carrier; constructing a plasmid carriers so as to obtain the bicistronic mRNA coexpression gene transporter. The gene transporter is not only pure and safe gene transporter but also realizes dual-gene coexpression in any combination, achieves the purpose of multi-gene coexpression through repeated utilization of IRES, and provides new thinking and route for improving the shape of dual or multiple gene control such as economic character.

The present invention relates to a retroviral vector which expresses a gene, e.g. for therapeutic use and/or of viral origin, under the translational control of an internal ribosomal entry site (IRES) resulting in the efficient translation of said gene.

Disclosed are a novel expression vector for efficient expression of recombinant proteins in mammalian cells, a mammalian cell transformed with the vector, and a method for production of the mammalian cell. The expression vector includes a gene expression regulatory site, and a gene encoding the protein downstream thereof, and an internal ribosome entry site further downstream thereof, and a gene encoding a glutamine synthetase further downstream thereof.

The invention provides a novel recombinant human blood coagulation factor VIII and a production method thereof. Particularly, the invention relates to reconstruction of a human blood coagulation factor VIII gene. A novel expression vector containing a weakened internal ribosome entry site (IRES for short) sequence and double screening marks is constructed. The vector can be used for high-efficiency recombinant expression of the human blood coagulation factor VIII, so that the expression of the vector in a mammalian cell expression system is increased.