7 results about "RNA polymerase III" patented technology

The application relates to an anti-apoptosis baculovirus expression vector, which realizes a broad-spectrum anti-apoptosis effect by expressing siRNA targeting a Sf-caspase-1 and Tn-caspase-1 common sequence of Spodoptera frugiperda and Trichoplusia ni insect cells through the vector. The baculovirus vector contains a specific DNA sequence, the DNA sequence contains an siRNA sequence targeting the Sf-caspase-1 and Tn-caspase-1 common sequence transcribed by an RNA polymerase III promoter; the recombinant virus of the vector can express double-stranded small RNA in a host cell, silences the caspase-1 encoded by the host cell through an RNA interference pathway, thereby inhibiting the apoptosis of the host cell, and significantly improving the expression level of an exogenous protein. The application can be used for industrialized production of protein preparations and vaccines.

The invention provides shRNA (short hairpin Ribonucleic Acid) molecules and application thereof in gene engineering and cell therapy. The multiple shRNA combination comprises three transcription units on the same vector, and the three transcription units can be used for respectively silencing target gene combinations such as TIGIT / CISH / HIF1A or TIM3 / TIPE2 / FAS and the like. Each unit comprises an RNA polymerase III promoter, an shRNA coding sequence and a termination signal, and the three promoters are different from one another and are selected from hU6, mU6, hH1 and the like. According to the design, homologous recombination is effectively avoided, and efficient, stable and lasting synchronous silencing of the three genes is achieved. The combination can also be connected with an overexpression unit containing CXCR2 and an IL-15R alpha / IL-15 fusion protein coding sequence to the same vector. Immune cells modified by the system have obviously enhanced survival, proliferation, migration and killing capabilities in a tumor microenvironment, and can be used for preparing drugs for treating diseases such as tumors.

The invention relates to a multiple shRNA combination and application thereof, and belongs to the technical field of biology. The combination comprises a first transcription unit, a second transcription unit and a third transcription unit which are located on the same expression vector and respectively silence three different target genes in a targeted manner. Each transcription unit comprises an RNA (Ribonucleic Acid) polymerase III promoter, an shRNA (Short Hairpin Ribonucleic Acid) coding sequence and a transcription termination signal; and promoters for driving the three transcription units are different from one another and are selected from any three of hU6, mU6, hH1, h7SK, mini-hU6, H1-7SK and hH1-core. According to the technology, simultaneous efficient, stable and lasting silencing of three target genes is realized by avoiding homologous recombination, nucleic acid constructs and expression vectors can be constructed and introduced into immune cells, the functions of the immune cells are effectively enhanced, and the method has application prospects in the fields of treatment of tumors, autoimmune diseases, senescence-related diseases and the like.

The invention relates to a quadruple shRNA (short hairpin ribonucleic acid) combination and application thereof. The combination comprises a first transcription unit, a second transcription unit, a third transcription unit and a fourth transcription unit which are located on the same expression vector and respectively silence four different target genes in a targeted manner. Each transcription unit comprises an RNA (Ribonucleic Acid) polymerase III promoter, an shRNA (Short Hairpin Ribonucleic Acid) coding sequence and a transcription termination signal; the promoters for driving the four transcription units are different from one another and are all selected from a promoter group consisting of hU6, mU6, hH1, h7SK, minihU6, H1-7SK and hH1-core; and the four transcription units are connected in series in an end-to-end manner. According to the design, by optimizing combination and arrangement of the promoters, expression interference and homologous recombination are effectively avoided, and efficient, stable, balanced and lasting silencing of the four target genes is achieved at the same time.

An adeno-associated virus (AAV) vector for inserting a desired nucleic acid into a nucleic acid in a cell, wherein the nucleic acid in the cell comprises a region consisting of a first nucleotide sequence and a region consisting of a second nucleotide sequence in order in a direction from a 5′ end to a 3′ end, wherein the vector comprises a first gRNA target sequence, a region consisting of a first nucleotide sequence, the desired nucleic acid, a region consisting of a second nucleotide sequence, a second gRNA target sequence, a cell-specific promoter, a sequence encoding a Cas9 nuclease, an RNA polymerase III promoter, a sequence encoding a first gRNA recognizing the first gRNA target sequence and a sequence encoding a second gRNA recognizing the second gRNA target sequence, wherein the vector yields a nucleic acid fragment comprising a region consisting of a first nucleotide sequence, the desired nucleic acid and the region consisting of the second nucleotide sequence by the Cas9 nuclease, wherein a first nucleotide sequence in the nucleic acid in the cell and a first nucleotide sequence in the vector are linked by a microhomology-mediated joining and a second nucleotide sequence in the nucleic acid in the cell and a second nucleotide sequence in the vector are linked by a microhomology-mediated joining, thereby inserting the desired nucleic acid between the region consisting of the first nucleotide sequence and the region consisting of the second nucleotide sequence in the nucleic acid in the cell.

The invention discloses an oil palm U3 promoter and application thereof, and belongs to the technical field of biology. The nucleotide sequence of the promoter is shown as SEQ ID NO.1. According to the invention, an upstream 400bp sequence of a U3 snRNA gene sequence is compared and intercepted in an oil palm genome for the first time, an oil palm RNA polymerase III type promoter-oil palm endogenous U3 snRNA promoter EgU3-1 is synthesized and obtained, and the promoter has efficient transcriptional activity and can drive the expression of downstream tracrRNA. The EgU3-1 promoter disclosed by the invention can be used for efficiently driving transcription of sgRNA in a gene editing carrier to obtain a large number of transcripts of the sgRNA, and an endogenous U3 promoter with high transcriptional activity is provided for efficient gene editing of oil palm.

The invention discloses a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9) gene editing system, a construction method of the CRISPR / Cas9 gene editing system and application of the CRISPR / Cas9 gene editing system in industrial strains of high-yield CPC cephalosporium acremonium, and the CRISPR / Cas9 gene editing system is a skeleton plasmid pCas9-sg and comprises a Cas9 gene, a functional sgRNA gene, a promoter pMBF, a promoter pTRPC, a constitutive RNA polymerase III type promoter pU6 and a terminator TTRPC hygromycin resistance selection marker hph. The system is high in efficiency, high in universality and easy to operate, and can be quickly applied to genetic modification of existing cephalosporium acremonium high-yield industrial strains. According to the method, efficient gene editing is realized in the high-yield CPC cephalosporium acremonium industrial strain for the first time, and the method is expected to be further used for modifying the cephalosporium acremonium industrial strain, shortening the time for genetic modification and upgrading of the strain, and providing powerful guarantee for improving the yield and purity of cephalosporin and increasing economic benefits.