94 results about "U6 promoter" patented technology

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 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 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 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 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 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 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.