757 results about "Insertion" patented technology

A method of transferring a gene to vertebrate cells is disclosed. The method comprises the steps of: (a) providing microprojectiles, the microprojectiles carrying polynucleic acid sequences, the sequences comprising, in the 5' to 3' direction, a regulatory sequence operable in the tissue cells and a gene positioned downstream of the regulatory sequence and under the transcriptional control thereof; and (b) accelerating the microprojectiles at the cells, with the microprojectiles contacting the cells at a speed sufficient to penetrate the cells and deposit the polynucleic acid sequences therein. Preferably, the target cells reside in situ in the animal subject when they are transformed. Preferred target cells are dermis or hypodermis cells, and preferred genes for insertion into the target cells are genes which code for proteins or peptides which produce a physiological response in the animal subject.

The invention discloses a method for site-specific mutagenesis of medicago sativa genes by employing a CRISPR / Cas9 system. First of all, a binary expression vector MsCRISPR / Cas9 is constructed; the vector is designed and constructed for a target gene; then, agrobacterium tumefaciens-mediated transformation is used to achieve site-specific mutagenesis of a specific gene, and a mutant transformed plant with the target gene knocked out is obtained by screening; the method uses the CRISPR / Cas9 technology for cross-pollination autotetraploid plant medicago sativa for the first time, and obtains a mutant plant; a MtU6 promoter is used for the first time to initiate sgRNA transcription in the medicago sativa, thereby improving the transcription efficiency; the mutation rate can reach 52%; the method realizes direct mutation on the coding sequence of the gene, and opens up a new field for gene editing in the medicago sativa, laying a technical foundation for simultaneous silencing of a plurality of genes, deletion of large chromosome fragments, precise insertion of foreign genes into targets, gene regulation and the like.

The invention relates to a method for achieving HMGCR gene knockout based on the CRISPR / Cas9 technology. The method is characterized in that two CRISPR / Cas9 target sequence aiming at the HMGCR gene isdesigned, a gRNA single chain is synthesized in vitro, annealing is performed to obtain two gRNA double-chain DNA target insertion fragments, the insertion fragments are inserted into PX459 (pSpCas9(BB)-2A-Puro)V2.0 vectors to obtain the two different-locus plasmids of the target HMGCR gene; the two plasmids are transfected into PK15 cells, puromycin is used to process the cells, the processed cell genome DNA is extracted to perform PCR amplification, the PCR product is denatured, annealing is performed, and then T7E1 is used to perform HMGCR gene knockout identification. The method has the advantages that method can be used for analyzing the expression conditions of sequence and mRNA after the HMGCR gene knockout, whether an off-target phenomenon exists or not can be verified by using amethod combining PCR and T7E1 enzyme treatment, and accordingly the specificity based on target sequence HMGCR-gRNA can be determined; the method is applicable to cell and animal models to achieve fixed-point HMGCR gene knockout, has a reference value to the knockout of other genes, and is good in effect, simple, economical, short in time and the like.

The present invention provides a method for the targeted insertion of a nucleotide of interest into a specific chromosomal site within a plant cell. The method comprises the steps of: (a) providing a plant cell, the plant cell optionally but preferably having a heterologous target site on a chromosome thereof, wherein said target site is flanked by at least one recombination site; and then (b) transforming said plant cell with a transformation vector (e.g., with an Agrobacterium transformation vector) carrying a nucleotide sequence of interest, wherein said nucleotide sequence of interest is flanked by at least one recombination site that corresponds to the recombination sites of said target site, so that said nucleotide of interest is inserted into said chromosome at said target site (when a target site is employed).

The invention belongs to the field of crop biotechnology and in particular relates to a molecular marker of a rice-blast-resistant gene Pi2 and application of the molecular marker. The upstream and downstream molecular markers are based on specific insertion / deletion loci polymorphism of nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2. The two molecular markers are co-dominant molecular markers developed for close linkage regions on two sides of the functional gene Pi2, and have the advantages of being high in accuracy and capable of being widely applied to different breeding parents. By detecting the two molecular markers, whether a detected rice material has the rice-blast-resistant gene Pi2 or not can be accurately judged. By virtue of a molecular marker combination, whether a hybrid transferred progeny plant genome of a parent of the functional gene Pi2 and other parents has the functional gene Pi2 or not and the homozygous state of the functional gene Pi2 can be accurately detected, and the breeding efficiency of a rice-blast-resistant rice variety with the functional gene Pi2 is improved.

The invention relates to novel insertion sites useful for the integration of HIV DNA sequences into the MVA genome, and to the resulting recombinant MVA derivatives.

Disclosed are the uses of specific genes of the mevalonate and isoprenoid biosynthetic pathways, and of inactive gene sites (the pseudogene) to (1) enhance biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate and isoprenoid pathway derived products in the plastids of transgenic plants and microalgae, (2) create novel antibiotic resistant transgenic plants and microalgae, and (3) create a novel selection system and / or targeting sites for mediating the insertion of genetic material into plant and microalgae plastids. The specific polynucleotides to be used, solely or in any combination thereof, are publicly available from GeneBank and contain open reading frames having sequences that upon expression will produce active proteins with the following enzyme activities: (a) acetoacetyl CoA thiolase (EC 2.3.1.9), (b) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (EC 4.1.3.5), (c) HMG-CoA reductase (EC 1.1.1.34), (d) mevalonate kinase (EC 2.7.1.36), (e) phosphomevalonate kinase (EC 2.7.4.2), (f) mevalonate diphosphate decarboxylase (EC 4.1.1.33), (g) isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2), and (h) phytoene synthase (EC 2.5.1.32).

The amino acid sequence of a mutant which is obtained by introducing a novel mutation into a Pseudonocardia thermophila JCM3095-derived nitrile hydratase consisting of two types of heterogeneous subunits, and the base sequence of the gene are provided. The nitrile hydratase is modified by specifying the region to be modified in the stereostructure / amino acid sequence of the nitrile hydratase, and applying alteration such as substitution, insertion, deletion or the like, to the amino acids in the amino acid sequence which are corresponding to the amino acid residues forming the region. Also provided is a method for modifying an enzyme having a nitrile hydratase activity.

The invention discloses an SSR (simples sequence repeats) and InDel (insertion / deletion) molecular marker primer linked with brassica campestris orange head gene Br-or and an application of the molecular marker primer. The marker primer is obtained by the steps of: based on the genome DNA (deoxyribonucleic acid) of orange brassica campestris and normal white brassica campestris and F2S4 separation group as a template, carrying out polymorphism primer screening to orange head and white head single strain DNA mixed tank in F2S4 by using 480 pairs of SSR and InDel primer pairs, then analyzing single strain of F2S4 group, and screening the molecular marker linked with the brassica campestris orange head gene Br-or to finally obtain fifteen Br-SSR and three Br-InDel molecular markers linked with the Br-or gene, wherein a molecular genetic map of the brassica campestris orange head gene Br-or is created on the ninth link group (A09). According to the link analysis, the genetic distance of two markers most tightly linked with both sides of the Br-or gene are 0.11cM and 0.79cM; and the molecular marker primer has the advantages of being convenient for detection, stable in amplification, and high in repeatability and accuracy, and thus a basis is provided to the molecule assistant selective breeding of brassica campestris orange head character and the breeding process is accelerated.

The invention relates to a CRISPR / Cas12a gene editing system and application thereof. The invention comprehensively utilizes biochemical, molecular biology, cell biology and other methods to screen and identify novel LiCas12a protein with endonuclease activity from a plurality of different bacteria. Based on LiCas12a, a CRISPR / LiCas12a gene editing system with high editing efficiency and high specificity is established in eukaryotic cells and prokaryotic cells separately, and genetic manipulation such as knockout, insertion and point mutation of genes is achieved; a molecular mechanism that the mutation of a TERT gene promoter promotes the cell proliferation of bladder cancer and liver cancer is disclosed. The genetic editing system based on CRISPR-LiCas12a provides a more accurate and efficient gene editing method for research fields such as disease pathogenesis and metabolic engineering transformation.