111 results about "AURKA Gene" patented technology

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 invention discloses a sgRNA specifically targeting human RSPO2 gene in a CRISPR-Cas9 system and an activation method and application thereof, and belongs to the field of biotechnology. The CRISPR-Cas9 system specifically activates the expression of the human RSPO2 gene, and introduces the activity of human hepatic stellate cells to activate Wnt signaling pathway so as to significantly upregulate the expression of liver fibrosis markers alpha-SMA and Collagen I. The CRISPR-Cas9 system designed for RSPO2 gene targets can effectively activate the hepatic stellate cells, thereby providing an effective way for liver fibrosis research.

The present invention describes methods and probe composition for an automated FISH assay of a blood sample containing circulating tumor cells expressing the IGF-1R gene. The assay provides genetic analysis of suspect circulating tumor cells that have been identified after immunomagnetic selection and fluorescent labeling. Using unique, repeat-free probes to the IGF-1R locus and a chromosome 15 reference probe, cell lines expressing an aberrant number of IGF-1R and Chr 15 signals were detected, including one cell line with a low level of IGF-1R amplification. The ability to directly examine the genetic profile of IGF-1R on circulating tumor cells may provide an automated means for assessing disease and patient response to therapy.

The novel gene, Rasp-1, which is up-regulated in regenerating liver, is disclosed. The novel RASP-1 protein, which is encoded by the Rasp-1 gene, is also disclosed. In addition, antibodies against the Rasp-1 gene products are also disclosed. Furthermore, the use of Rasp-1 nucleic acid sequences, Rasp-1 gene products, and antibodies against Rasp-1 gene products in the treatment and diagnosis of liver disorders is also disclosed.

The invention discloses a kit for investigating polymorphism of an MTHFR (Methylene Tetrahydrofolate Reductase) and / or MTRR (Methylenetetrahydrofolate Reductase) gene on the basis of a Taqman-MGB probe. The kit comprises a primer group and a probe, wherein the primer group is at least one selected from the following three groups: primers of SEQ ID NO.1 and SEQ ID NO.4 for an MTHFR gene at a C677Tsite, primers of SEQ ID NO.7 and SEQ ID NO.10 for an MTRR gene at an A1298C site, primers of SEQ ID NO.13 and SEQ ID NO.14 for an MTRR gene at an A66G site, primers of SEQ ID NO.22 and SEQ ID NO.19, primers of SEQ ID NO.29 and SEQ ID NO.15, and primers of SEQ ID NO.33 and SEQ ID NO.30. The kit disclosed by the invention has the advantages that three mutation sites can be detected simultaneously, high sensitivity is achieved, plasma as low as 10copies can be accurately detected, and oral cavity swabs which are too long in preservation time or relatively low in concentration can be still accurately detected.

The invention discloses an application of CDC20 co-expression gene network as a glioma treatment target. The invention provides an application of an inhibitor in any of the following: (A1) preparing aproduct for treating glioma; (A2) treating glioma, wherein the inhibitor is a substance capable of reducing the expression activity and / or expression level of the following genes or encoded proteinsthereof: AURKA gene, CDC20 gene and / or KIF2C gene. The application proves that CDC20-M is a sensitive marker of genomic instability and poor prognosis in glioma, and finds potential glioma treatment targets in experimental analysis of main members of CDC20-M, and thus a new idea is provided for clinical diagnosis and treatment.

The invention discloses a multiplex PCR (Polymerase Chain Reaction) trapping primer group for a DMD (Duchenne Muscular Dystrophy) gene, a mutation detection kit for the DMD gene and a mutation detection method for the DMD gene. The inventors provide the multiplex PCR trapping primer aiming at the sequences of 79 exons and bilateral 50bp introns and 23 known intron pathogenic mutations of the DMD gene; moreover, the mutation detection method of the DMD gene is provided based on the primer group; the mutation detection method is high in detection sensitivity, good in accuracy, highly consistentin amplification efficiency and low in requirement on quality of a DNA (Deoxyribonucleic Acid) sample, and is used for solving the problem that the complete detection on the mutation information, particularly on loss of heterozygosity / repetition, of the DMD gene is difficultly carried out in one time by adopting a multiplex PCR trapping technique in the prior art.

The invention relates a PML gene and RARA gene detection probe and a preparation method thereof. The method comprises the following steps that at least one of RP11-832J18, CTD-2529B11, RP11-756N20 and RP11-1031J4 and a selected BAC cline for an RARA gene is at least one of CTD-2360L10, RP11-737D6, CTD-3087O22, RP11-48O10 and CTD-2134K5 are included; a plasmid DNA is obtained; labeling is performed. The invention further discloses a reagent kit containing the PML gene and RARA gene detection probe for detecting acute promyelocytic leukemia PML and RARA fusion genes. The optimal PML gene and RARA gene detection probe is obtained through screening, the signal counting row is accurate and rapid, and the result repeatability is good.

Belonging to the field of tumor gene therapy, the present invention provides an anti-tumor RNA interference plasmid-lipidosome-heparin complex targeting the human FAK and FLK1 genes. The RNA interference expression vector can express a RNA interference sequence aimed at a target site shown by SEQ ID NO:1 on the FAK gene and a RNA interference sequence aimed at a target site shown by SEQ ID NO:2 on the FLK1 gene in the body. The FAK and PLK1 dual-gene RNA interference plasmid prepared by the present invention can effectively inhibit the growth of lung cancer cells and induce the lung cancer cells to be apoptosed. A tumor inhibition experiment conducted in the body also indicates that the FAK and PLK1 dual-gene RNA interference plasmid-lipidosome-heparin complex can notably inhibit the growth of manifold tumors and prolong the lifetime of tumor-bearing mice. The product of the anti-tumor RNA interference plasmid-lipidosome-heparin complex has obvious efficacy and can reduce dosage and improve the life quality of patients, thus having a good application prospect.

The invention relates to a recombined lentivirus vector aiming at PKC gamma gene RNA interference, construction thereof and application thereof. A lentivirus vector of ShRNA aiming at PKC gamma gene is constructed by experiments; a synthesized DNA fragment aiming at the ShRNA is mediated through the lentivirus vector, and transfects a 293T cell with pHelper 1.0 vectors and pHelper 2.0 vectors for culturing; and after the recombined lentivirus vector is acquired, a target cell is transfected to realize RNA interference aiming at the PKC gamma gene. The self-inactivated third generation lentivirus (SIN) is adopted, and the recombined lentivirus vector has the advantages of safety and reliability, unseparated cell infection, long-term expression for the target gene integrated to a target cell gene group, small immunity reaction and the like, and is an ideal vector. The lentivirus vector has interference effect on rat primary culture neuron over 85 percent, so that the recombined lentivirus vector can become a powerful tool for treating gene of chronic neuropathic pain.

The present invention provides one small interference ribonucleic acid (SiRNA) as the active component for antitumor medicine to kill tumor cell and for epidermal growth factor Cripto gene mRNA. Its nucleotide sequence has one negative chain and one nucleotide mutant without homology to known human gene. The SiRNA can inhibit the expression of human colon cancer LS-174T cell Cripto gene effectively via intracellular RISC, and is favorable to treating colon cancer and other malignant tumor with up expressing Cripto gene. Therefore, the SiRNA may be used in medicine for treating malignant tumor and relevant disease with up expressing Cripto gene.

The invention discloses a human embryonic stem cell line capable of controlling gene expression and application thereof. The human embryonic stem cell line capable of controlling gene expression comprises a controlled expression exogenous fusion protein system and at least one guide RNA, and the controlled expression exogenous fusion protein system comprises a tetracycline inducible expression system and fusion protein formed by Cas9 protein and p300 core domain with transcriptional activation activity; each guide RNA is a 14bp short data sequence of a targeting gene promoter region or a 20bplong data sequence of a genome region. Accordingly, expression of Cas9-p300 in human embryonic stem cells can be controlled in real time, the specific gene activation and cutting double functions of human embryonic stem cell differentiation in different stages are achieved, and application of the human embryonic stem cells in the fields of medicine science and lift science is greatly extended.

The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of Bach1 gene expression and / or activity, and / or modulate a Bach1 gene expression pathway. Specifically, the invention relates to double-stranded nucleic acid molecules including small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules that are capable of mediating or that mediate RNA interference (RNAi) against Bach1 gene expression.

The invention belongs to the technical field of biology, and provides a method for constructing and identifying systemic Plin1 gene knockout animal model. The construction method comprises the following steps: designing sgRNA sequences aiming at a front sequence and a rear sequence of an exon 2 of a Plin1 gene, constructing a Plin1 gene knockout vector, and carrying out in-vitro transcription to obtain sgRNA; and mixing the sgRNA with Cas9 protein, and then performing microinjection into fertilized eggs of a mouse to obtain a systemic Plin1 gene knockout mouse. The method has the advantages that: a pair of the sgRNA sequences with the highest score is screened out, and the success rate of the project is guaranteed; the large-fragment knockout of the exon 2 of the Plin1 gene is realized, and the knockout efficiency is improved; and three primers are designed for the knockout area, various genotypes of mice are successfully identified, and the situation that two primers may not distinguish genotypes when large fragments are knocked out is avoided. The invention provides the convenient, reliable and economic animal model and a good foundation for researching the biological function ofPLIN1 and the pathogenesis of obesity-related metabolic diseases.

The invention discloses a specificity primer and a liquid phase chip for SNP detection of CYP4F2 gene. The liquid phase chip comprises an ASPE primer formed by the specificity primer aiming at a target gene SNP locus of the tag sequence on the 5' end and 3' end, wherein the specificity primer is SEQ ID NO.13 and SEQ ID NO.14 aiming at the G1297A SNP locus and / or SEQ ID NO.15 and SEQ ID NO.16 aiming at the T34G SNP locus, a microsphere and an amplification primer. The invention also provides a liquid phase chip for SNP detection of CYP4F2 and EPHX1 genes, which comprises the corresponding compositions of the liquid phase chip for the SNP detection of the CYP4F2 and EPHX1 genes, an ASPE primer pair aiming at the G357A, G19512990A, T337C and / or A416G SNP locus of the EPHX1 gene, microsphere and amplification primer. The detection liquid phase chip has an excellent signal-noise ratio, can avoid cross reaction and can realize the parallel detection of a plurality of SNP loca.

The invention relates to the field of molecular biology, genetic engineering technology and biological medicine, in particular to a small interfering RNA (ribonucleic acid), a short hairpin RNA and a recombined carrier for SOX4 gene targets, and application of the recombined carrier. The small interfering RNA comprises a positive-sense strand shown as SEQ ID NO.1 and an antisense strand shown as SEQ ID NO.2. The short hairpin RNA comprises a positive-sense strand shown as SEQ ID NO.3 and an antisense strand shown as SEQ ID NO.4. The recombined carrier comprising the short hairpin RNA can be transcribed in a host cell to obtain SOX4-shRNA, the small interfering RNA can be generated after the SOX4-shRNA is processed by the host cell, a messenger RNA of SOX4 is targeted, expression of the SOX4 is reduced, the gene silencing effect is caused, multiplication of esophageal squamous carcinoma cells is inhibited effectively, and aging of the esophageal squamous carcinoma cells induced by chemotherapeutic adriamycin is promoted, so that the recombined carrier has application value in tumor resistance, and the foundation is laid for further research on the function of the SOX4 in aging and escaping of tumor cells and for research and development of targeted SOX4 drugs.

The invention provides a kit and a method for detecting NTRK gene fusion. The kit includes at least one of four tubes of reagents, the 4 tubes of the reagents contain specific primers and probes designed for 12 fusion types of a NTRK gene, one kind of NTRK1 fusion, one kind of NTRK2 fusion and one kind of NTRK3 fusion are detected in each tube, interference between the primers of the same gene canbe excluded, thus the stability of a reaction system is greatly improved, the detection sensitivity and specificity are improved, and the probability of occurrence of false positives is decreased.

The invention provides primers for detecting MPL gene mutation, and the primers include a forward primer and a backward primer for MPL gene exon 10. The invention belongs to the technical field of biological detection, and the primers provided by the invention can specifically detect MPL gene exon 10 mutation, and the specificity of the primers is good, and the accuracy is high, and the efficiency of the detection is increased.

The invention discloses a method for detecting CYP21A2 gene mutation related to congenital adrenal hyperplasia, a primer and a kit. The primer and the kit respectively comprise an amplification primerand a sequencing primer aiming at all exons of a CYP21A2 gene. Based on PCR amplification and Sanger sequencing, the mutation condition of the mutation site of the CYP21A2 gene related to the congenital adrenal hyperplasia can be quickly detected.

The present invention concerns methods and reagents useful in modulating transcription factor gene expression in a variety of applications, including methods of use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), and doublestranded RNA (dsRNA) molecules capable of mediating RNA interference (RNAi) against E2F1 gene expression, useful in the treatment of cell cycle disorders, inflammatory conditions, reproductive disorders, cancers and any other condition that responds to modulation of E2F1 expression and / or activity.

The invention relates to an MTHFR gene polymorphism detection primer. The MTHFR gene polymorphism detection primer comprises a forward primer used when an MTHFR gene c.1298 locus is in an A condition, a reverse primer used when the MTHFR gene c.1298 locus is in the A condition, a forward primer used when the MTHFR gene c.1298 locus is in a C condition, a reverse primer used when the MTHFR gene c.1298 locus is in the C condition, a forward primer used when an MTHFR gene c.677 locus is in the C condition, a reverse primer used when the MTHFR gene c.677 locus is in the C condition, a forward primer used when an MTHFR gene c.677 locus is in the T condition, a reverse primer used when the MTHFR gene c.677 locus is in the T condition, a forward primer for detecting a beta-actin gene and a reverse primer for detecting the beta-actin gene. An MTHFR gene polymorphism detection kit is rapid and convenient to detect, high in sensitivity, high in accuracy rate and low in cost.

The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of stromal cell-derived factor-1 (SDF-1) gene expression and / or activity. The present invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and / or activity of genes involved in SDF-1 gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating or that mediate RNA interference (RNAi) against SDF-1 gene expression. Such small nucleic acid molecules are useful, for example, in providing compositions for treatment of traits, diseases and conditions that can respond to modulation of SDF-1 expression in a subject, such as ocular disease, cancer and proliferative diseases and any other disease, condition, trait or indication that can respond to the level of SDF-1 in a cell or tissue.

The invention relates to a method of knocking out spinal muscular atrophy SMN genes and a cell model. The cell model is constructed by adopting the following steps: (1) constructing and designing zinc finger nuclease plasmids for knocking out the SMN genes; (2) transferring the constructed ZFN plasmids for knocking out the SMN genes into mammalian cells by using a lipidosome method; (3) detecting the effectiveness of the ZFN gene knockout by using a genome PCR product of a ZFN peculiar knockout site and DNA sequence determination; (4) specific to the cells, the ZFN genes of which are effectively knocked out, carrying out monoclonal separation and identifying cell knockout types one by one; (5) carrying out mRNA level identification on gene knockout cell strains; (6) carrying out protein expression level identification on the gene knockout cell strains. The spinal muscular atrophy pathology cell model can be used for research of SMA pathogenesis; medicines can be screened by detecting the level of protein expression of SMN compounds in the cell model.

The invention discloses a U1-snRNA for repairing TPP1 gene splicing defects and a vector containing a base sequence for expressing the U1-snRNA. The base sequence for expressing the U1-snRNA sequentially comprises a U1-snRNA initial basic group, a sequence complemented with a TPP1 gene Exon12 donor splicing site or a downstream intron sequence target and a U1-snRNA downstream sequence, and the U1-snRNA downstream sequence is as shown in SEQ ID NO.20. The U1-snRNA is from a nucleic acid molecule prepared according to a TPP1 gene donor splicing site or donor splicing site downstream intron sequence. By the aid of a U1-snRNA technique, U1-snRNA of a target gene serves as a target medicine, and repairing is implemented in the splicing process of Pre-mRNA, so that aberrant mRNA expression caused by splicing site mutation is effectively restrained. The U1-snRNA has the advantages of high specificity, high efficiency and small side effect, can make up for the deficiency of current treatment means of diseases caused by aberrant splicing and possibly serves as a new method for treating specific types of diseases in the near future.

The invention relates to the technical field of genetic engineering, and especially relates to a sgRNA for targeted knockout of a FRZB gene, a porcine embryo fibroblast cell line for knocking out theFRZB gene and applications of the fibroblast cell line. The provided double sgRNA can cut two loci on the first exon of the FRZB gene, then after the sgRNA is constructed into an expression vector, then porcine embryo fibroblast cells are transfected through an optimized electro-transformation system, and a flow cytometer is used to sort positive monoclonal cells with high purity, so that the FRZBknocked-out cell line can be obtained through genome identification and screening. Thus, the problems of low transfection efficiency, low targeting efficiency and low monoclonal purity during the construction of knockout cell lines can be solved; and simple operation can be realized, and the edited cell line with longer fragment deletion can be obtained. The related cell line can be used as the donor of nuclear transfer of somatic cells for the preparation of transgenic swine, so that good research tools can be provided for further exploring the biological functions of the FRZB gene.

The invention provides a kit for detecting lung cancer susceptibility genes. The kit comprises a primer aiming at a single nucleotide polymorphism site rs4488809 (T-C) on a TP63 gene, a primer aiming at a single nucleotide polymorphism site rs36600 (T-C) on an MTMR3 gene, and a primer aiming at a single nucleotide polymorphism site rs2395185 (G-T) on an HLA gene.

The invention provides primers and reagent kits for detecting CYP21 gene mutation and an application of the primer and reagent kit. The primers comprise a specific primer for an overall length sequence of a CYP21A2 gene, a specific primer with 30kb deletion and mutation of a CYP21 authenticity gene and a specific primer of recombination / fusion mutation of the CYP21 authenticity gene. The constructed reagent kits and primers are small in quantity, detection of various mutation types of the CYP21 gene can be completed once, the process is simple, the results are high in precision, and the primers and the reagent kits have broad application prospects in the field of diagnosis of congenital adrenal hyperplasia.

The invention relates to a TERC gene and MYC gene detection probe and a preparation method thereof. The method comprises the following steps that at least one CTD-3214J12, RP11-816J6 and RP11-778I3 is selected as a BAC clone for a TERC gene, or at least one of RP11-3K16, CTD-2582E13 and RP11-886J24 is selected as a BAC clone; and / or CTD-2511O8 is selected as a BAC clone for an MYC gene, or at least one of RP11-440N18 and CTD-2205H22 is selected as the BAC clone; a plasmid DNA is obtained; labeling is performed. The invention further discloses a reagent kit containing the TERC gene and MYC gene detection probe. The optimal TERC gene and MYC gene detection probe is obtained through screening, signal counting row is accurate and rapid, and the result repeatability is good.

The present invention discloses a construction method of a SIRT1 gene knockout IPEC-J2 cell line. The construction method comprises the following steps: (1) a gRNA probe is designed and synthesized for a first exon of a SIRT1 gene, a base sequence of gRNA is shown as SEQ ID NO.1, and a reverse complement sequence is shown as SEQ ID NO.2; (2) an inverted complementary double-stranded gRNA probe after annealing is cloned into a pSpCas9-2A-PuroV2.0 vector shown in figure 2, and a plasmid simultaneously expressing the sgRNA as shown in the SEQ ID NO.1 and Cas9 proteins is constructed; (3) the plasmid constructed in the step (2) is transfected into IPEC-J2 cells; and (4) a monoclonal mutant cell line is screened from the transfected IPEC-J2 cells in the step (3) to obtain the SIRT1 gene knockout IPEC-J2 cell line, and compared with a wild-type cell line, the SIRT1 gene knockout IPEC-J2 cell line lacks 5 bases in the first exon of the SIRT1 gene. The SIRT1 gene knockout IPEC-J2 cell line isfirstly constructed, the stable monoclonal SIRT1 gene knockout IPEC-J2 homozygous cell line is screened, and the SIRT1 gene knockout IPEC-J2 cell line can be used as a cell model for studying intestinal oxidative stress of piglets.