40 results about "AURKA Gene" patented technology

The invention discloses a gene engineering exosome and a slow release method and application thereof, the gene engineering exosome is derived from human adipose-derived stem cells, si-tgfbr2 is loaded in the gene engineering exosome, the si-tgfbr2 is specific siRNA aiming at a Tgfbr2 gene, and the gene engineering exosome can regulate and control proliferation of VSMCs, inhibit transformation of the VSMCs from a contraction phenotype to a synthetic phenotype when the VSMCs are stimulated, and inhibit the growth of the VSMCs. According to the present invention, the gene engineering exosome can be used to maintain the normal structure and the function of the blood vessel, and the sustained release effect of the content in the gene engineering exosome can be achieved by wrapping the gene engineering exosome in the PHA biodegradable material, such that the local treatment concentration can be easily stabilized, the drug administration frequency can be easily reduced, the lasting time of the treatment effect can be easily prolonged, and the treatment gene can be effectively delivered to the target cell; the treatment effect is improved; when being used for modifying a small-caliber artificial blood vessel, the small-caliber artificial blood vessel can be improved in biocompatibility and functionality, and can be prevented from being implanted into the blood vessel for remodeling unbenign hyperplasia stenosis; the invention not only provides a new thought and method for the treatment of vascular diseases, but also has important clinical application value and popularization prospect.

Disclosed herein are nucleic acid compositions and methods of use. The nucleic acid compositions may have a therapeutic nucleic acid sequence operably linked to a nuclear targeting sequence that increases expression of the therapeutic nucleic acid in a cell by at least 1.25 fold; at least 80% sequence identity to SEQ ID NO: 6; or a first regulatory element comprising a promoter sequence operably linked to a hemoglobin subunit gamma intron (hBGi) sequence, and a second regulatory element comprising a woodchuck hepatitis posttranscriptional regulatory element (WPRE) sequence. The nucleic acid compositions with these features may enhance therapeutic nucleic acid transfection and expression. Also disclosed herein are transgenes optimized for gene therapy applications, including novel FVIII transgene sequences, in which the B domain may be non-naturally occurring the A1 and / or A3 domain may include at least one amino acid substitution.

The invention discloses a construction method of a glycogen accumulation disease type 3 (GSD III) zebrafish F0-generation model and application of the model in drug screening, sgRNA combination and Cas9 protein are mixed, the mixture is jointly injected into a single cell stage embryo of wild type zebrafish, and the F0-generation zebrafish model generating diallele mutation is obtained through cultivation. The sgRNA combination comprises a first targeting sequence and a second targeting sequence aiming at the agla gene, and a third targeting sequence and a fourth targeting sequence aiming at the aglb gene. The technical barrier that the construction period of a traditional GSD III zebrafish double-knockout homozygous strain is as long as 6-12 months is broken through, through the screened high-activity sgRNA combination and the adoption of a high-concentration Cas9 protein co-injection strategy in the single cell period, the extremely high biallele mutation rate is achieved in the F0 generation, and the high-concentration Cas9 protein co-injection strategy is adopted in the single cell period. The defects that the traditional F0 generation is high in chimeric degree and unstable in phenotype are effectively overcome.

The invention belongs to the technical field of gene editing and disease model construction, and particularly relates to a construction method and application of a Wilson disease animal model. Aiming at a large fragment deletion mutation type which exists clinically in Wilson disease but lacks a corresponding animal model, a CRISPR / Cas9 gene editing technology is utilized, a pair of sgRNAs is specifically designed, an eighth exon region of a mouse Atp7b gene is precisely cut and deleted, and a WD mouse model with deletion mutation (c.2333340delGACGGTGG) of eight basic groups of the eighth exon of the Atp7b gene is successfully constructed. The method can be used for research on pathogenesis of WD, screening of novel diagnostic markers and evaluation of curative effect of therapeutic drugs, is particularly suitable for preclinical evaluation of gene therapy strategies and development of adjuvant therapeutic drugs of targeted NLRP3 inflammasomes, and has great scientific research value and clinical transformation prospect.

The present invention discloses a construction method and application of a mouse conditional inducible Hmox1 gene knockout model, and belongs to the field of biotechnology. The mouse conditional inducible Hmox1 gene knockout model provided by the present invention is constructed by the following steps: constructing a homologous recombination DNA vector; obtaining Cas9mRNA and gRNA for the Hmox1 gene by in vitro transcription; injecting the homologous recombination DNA vector, Cas9mRNA and gRNA into the fertilized egg of the mouse; cultivating to obtain conditional gene knockout mice; identifying the genotype by sequencing and nucleic acid gel electrophoresis; the obtained conditional knockout mice are hybridized with other tissue-specific Cre-expressing mice to obtain corresponding tissue or cell conditional knockout mice, thereby realizing precise research on specific tissues or cell types; and proving that Hmox1 inducible conditional knockout significantly aggravates acute lung injury. The present invention can overcome the difficulties of embryonic lethality of Hmox1 systemic gene knockout mice in the prior art and the inability to construct a mouse model, and assist in the study of Hmox1 in diseases.

The invention discloses a method for preparing a CD163 gene editing pig based on a CRISPR (clustered regularly interspaced short palindromic repeats) / Gs12-7MAX system. The method specifically comprises two editing strategies: one is to edit a sixth intron and a seventh intron of a CD163 gene to realize accurate deletion of a seventh exon so as to obtain a porcine fibroblast monoclonal cell with homozygous deletion of the seventh exon; and 2, editing aiming at a seventh exon of the CD163 gene, and inducing the exon to generate frame-shift mutation to obtain a gene editing pig. The technical system established by the invention has the advantages of high editing efficiency, strong accuracy and simplified operation process, provides a new effective strategy for prevention and control of PRRSV (porcine reproductive and respiratory syndrome), and has important popularization and application values in creation of new materials for pig disease-resistant breeding.

The invention relates to a primer and probe composition, a kit and a method for detecting a polymorphic site of a folate metabolism key enzyme gene. The primer and probe composition comprises at least one of primers and probes aiming at a C677T site of an MTHFR (Methylene Tetrahydrofolate Reductase) gene, an A1298C site of the MTHFR gene and an A66G site of an MTRR (Methylene Tetrahydrofolate Reductase) gene. The primer and the probe can independently realize detection and can be combined in the same reaction system and reaction program for multiple PCR amplification, the primer and the probe aiming at different polymorphic sites do not interfere with each other, Tm values of amplification products aiming at different genotypes of the three polymorphic sites are obviously different, and the Tm values of the amplification products aiming at different genotypes of the three polymorphic sites are obviously different. Therefore, simultaneous detection of the three gene polymorphic sites can be realized, and the method has the advantages of short detection time, high detection sensitivity, good specificity and high accuracy.

This invention relates to a kit and method for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases. The kit includes a library hybridization reagent and a probe set for detecting XLIRD pathogenic genes, including capture probes that can simultaneously and specifically capture the RPGR, RP2, RS1, and CHM pathogenic genes. The capture probes for capturing the RPGR pathogenic gene include probes with sequences as shown in SEQ ID NO. 1-96, and capture probes targeting the high-GC region of the RPGR gene with sequences as shown in SEQ ID NO. 24-55. This invention targets the above four pathogenic genes, comprehensively analyzes their variation profiles and sequence characteristics, optimizes the amplification region range, and designs a high-density capture probe set including exons, introns, UTR regions, and upstream and downstream 20kb regions. Specifically targeting the high-GC content region of exon 15 of the RPGR gene, the probe sequence and capture strategy are adjusted to achieve efficient capture of the high-GC region. During the library PCR enrichment stage, the amplification enzymes in the system are screened and optimized. Combined with the probe set, multiple amplification enzyme systems are used, and finally, the most suitable amplification enzyme combination for this gene system is optimized.

The invention provides a method for constructing an ANXA2 gene knockout PK-15 cell line based on a CRISPR / Cas9 technology. The method comprises the steps of sgRNA design, vector construction, cell transfection, monoclonal screening, gene knockout effect verification and the like. Specific sgRNA aiming at an ANXA2 gene is designed and synthesized on the basis of a swine-derived AnxA2 gene sequence, a recombinant plasmid is constructed by taking a plasmid pSpCas9 (BB) as a carrier, a PK-15 cell is transfected, stable expression cells are obtained through puromycin screening and then are monoclonal, the obtained monoclonal cells are verified through methods such as genome DNA sequencing, western blotting and DNA gel electrophoresis, and the detection result shows that the ANXA2 gene sequence can be used for detecting the ANXA2 gene. The PK-15 cell line with the ANXA2 gene knocked out, which is stably expressed, is successfully obtained. The ANXA2 protein expression of the cell line is obviously reduced, the gene sequence is subjected to base mutation, the cell activity is not influenced, the cell line can be used for researching the action mechanism of ANXA2 in virus invasion and replication, and an important tool is provided for virology research.

Disclosed herein are nucleic acid compositions and methods of use. The nucleic acid compositions may have a therapeutic nucleic acid sequence operably linked to a nuclear targeting sequence that increases expression of the therapeutic nucleic acid in a cell by at least 1.25 fold; at least 80% sequence identity to SEQ ID NO: 6; or a first regulatory element comprising a promoter sequence operably linked to a hemoglobin subunit gamma intron (hBGi) sequence, and a second regulatory element comprising a woodchuck hepatitis posttranscriptional regulatory element (WPRE) sequence. The nucleic acid compositions with these features may enhance therapeutic nucleic acid transfection and expression. Also disclosed herein are transgenes optimized for gene therapy applications, including novel FVIII transgene sequences, in which the B domain may be non-naturally occurring the A1 and / or A3 domain may include at least one amino acid substitution.

The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the CIDEB gene, as well as methods of inhibiting expression of CIDEB, and methods of treating subjects that would benefit from reduction in expression of CIDEB, such as subjects having a CIDEB-associated disease, disorder, or condition, using such dsRNA compositions.

The application discloses a kit for detecting LINC01480 gene expression and a preparation method and application thereof, relates to the field of gene expression kits, and is used for detecting LINC01480 gene expression and comprises a specific primer pair for the LINC01480 gene, a specific primer pair for a reference gene GAPDH, a positive control lentivirus vector containing a LINC01480 gene segment, a negative control lentivirus vector and an RT-qPCR reaction system. The application non-invasively monitors MRD of MM patients after treatment by detecting LINC01480 gene expression, so as to improve the convenience and accuracy of index monitoring of MRD of the MM patients after treatment.

The application discloses a gene editing system for constructing a GP130 gene mutation gastric cancer model pig nuclear transfer donor cell and application thereof. The gene editing system comprises a high-efficiency Cas9 protein prepared according to the method of the application, a high-efficiency target gRNA for the GP130 gene screened, and a single-chain Donor DNA containing a GP130 mutation site, and the optimal use amount ratio of the components of the system is optimized, and finally the single-cell clone ratio of the target site point mutation is 22.5%, which is much higher than the conventional point mutation efficiency (<5%).

The application discloses a construction method of a glycogen storage disease type 3 (GSD III) zebrafish F0 generation model and application thereof in drug screening, mixes sgRNA combinations with Cas9 protein, and jointly injects into single-cell period embryos of wild type zebrafish to cultivate and obtain F0 generation zebrafish models with double allelic mutations. The sgRNA combinations comprise a first targeting sequence and a second targeting sequence for an agla gene, and a third targeting sequence and a fourth targeting sequence for an aglb gene. The application breaks through the technical barrier that a traditional GSD III zebrafish double knockout homozygous strain construction cycle lasts for 6-12 months, through screening of high-activity sgRNA combinations and a high-concentration Cas9 protein co-injection strategy in the single-cell period, the application realizes a very high double allelic mutation rate in the F0 generation, and effectively overcomes the defects of high chimerism and unstable phenotype in the traditional F0 generation.

The invention discloses application of a Circ-arRNA base editor in a DFNA15 deafness mouse model, and belongs to the technical field of biomedicine. The base editor according to the present invention comprises: a base sequence encoding ribozyme having autonomous cleavage activity for promoting arRNA cyclization; rNA connexons for connecting the functional elements; and an arRNA sequence (targeting a POU4F3Q113 * mutation site), which is used for specific recognition and targeting binding of a POU4F3Q113 * transcript. Specifically, aiming at a c.C337T (p.Q113 *) mutation site transcript of a POU4F3 gene, a Circ-arRNA111nt-A3 editing tool with the length of 108 nucleotides (nt) is obtained through screening and optimization, and the tool realizes the optimal balance between the editing efficiency and the biological safety. Furthermore, a CAG-U6 dual promoter system is adopted to drive the Circ-arRNA expression, so that the mutant base editing efficiency is improved by 200%. The base editor disclosed by the invention has high efficiency and safety, not only provides a key technical basis for clinical treatment of DFNA15 hearing impairment, but also establishes a promotable methodological framework for gene therapy research of other hereditary point mutation diseases.

To provide a staple nucleic acid to TRPC6 gene.SOLUTION: Provided herein is an inhibitor of the expression of TRPC6 protein comprising a staple nucleic acid against the TRPC6 gene.SELECTED DRAWING: None

The invention relates to the technical field of molecular oncology and drug resistance mechanism research, and discloses an S232F site of acquired drug resistance of a fusion gene STRN3-RARA and application of the S232F site in ATRA. The product is used for detecting the sensitivity of a patient suffering from acute promyelocytic leukemia to all-transretinoic acid (ATRA) treatment or screening whether the patient suffering from acute promyelocytic leukemia is resistant to ATRA treatment, an S232F mutation site is an STRN3-RARA fusion gene coding protein region mutation site, and the nucleotide sequence of the STRN3-RARA fusion gene is shown as SEQ ID NO: 1. The S232F mutation site is located at the 974th nucleotide site of the nucleotide sequence, and the base C is mutated into T. According to the present invention, the S232F mutation site can be adopted as the drug resistance detection marker, the reagent or the kit can be designed according to the STRN3-RARA gene S232F mutation site so as to be used for clinical drug resistance detection, and the gene mutation state of the patient can be monitored so as to guide the personalized drug use.

The invention discloses an application of a phytanyl coenzyme A dioxygenase structural domain 1 in in-vitro reduction of an intracellular Tau protein level and a method for in-vitro reduction of an intracellular Tau protein expression level. According to the method, on the basis of early-stage research results aiming at PHYHD1 genes and proteins, effective overexpression of the PHYHD1 proteins in HEK-293 Tau cells capable of stably expressing Tau proteins is realized by preparing a transfection compound. The method can significantly reduce the Tau protein level by 33.6%, the process is simple, convenient and reliable, and the transfection operation does not have significant cytotoxicity. The method can be widely applied to the fields of AD basic research, drug screening, gene therapy strategy development and the like.

The invention relates to a kit and a method for detecting pathogenic genes of hereditary retinal degeneration diseases linked with X chromosomes, the kit comprises a library hybridization reagent, the reagent comprises a probe group for detecting XLIRDs pathogenic genes, and the probe group comprises capture probes capable of simultaneously and specifically capturing RPGR, RP2, RS1 and CHM pathogenic genes; the capture probe for capturing the RPGR pathogenic gene comprises a probe with a sequence shown as SEQ ID NO.1-96, and a capture probe for a high GC region of the RPGR gene, and the sequence of the capture probe is shown as SEQ ID NO.24-55. Aiming at the four pathogenic genes, variation spectrums and sequence characteristics of the four pathogenic genes are comprehensively analyzed, an amplification region range is optimized, and a high-density capture probe group containing exons, introns, UTR regions and upstream and downstream 20kb regions is designed. Particularly aiming at a high-GC-content region of a No.15 exon of the RPGR gene, a probe sequence and a capture strategy are adjusted to realize efficient capture of the high-GC-content region; in a library PCR enrichment stage, amplification enzymes in the system are screened and optimized, and an amplification enzyme combination most suitable for the gene system is finally optimized by combining a probe group and adopting a plurality of amplification enzyme systems.

The present invention provides a dual nucleic acid sequence for inhibiting melanoma growth and its application, as well as the use of a substance that reduces the expression levels of the ITK and MAT2B genes in the preparation of a drug for treating or preventing melanoma. The substance that reduces the expression levels of the ITK and MAT2B genes includes RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, and substances for implementing lentiviral infection or gene knockout targeting the ITK and MAT2B genes. The present invention provides the use of the ITK and MAT2B genes in the treatment of melanoma. By specifically interfering with the ITK and MAT2B genes, the malignant progression of melanoma can be effectively inhibited. The ITK and MAT2B genes can be used as targets for the treatment of melanoma and have good clinical application value.

The invention belongs to the technical field of mouse model construction, and particularly relates to a construction method of a chronic myelogenous leukemia mouse model with RUNX1 gene conditional knockout. The method comprises the following steps: hybridizing a RUNX1 gene conditional knockout mouse and an LYZ2-CreERT2 mouse to obtain an F1 generation, and carrying out DNA (Deoxyribose Nucleic Acid) identification and screening to obtain a double-transgenic mouse with the genotype of LYZ2-CreERT2 / RUNX1; then hybridizing the LYZ2-CreERT2 / RUNX1 double-transgenic mouse of the F1 generation with the SCL-tTA / Bcr-abl double-transgenic mouse to obtain an F2 generation, and carrying out DNA (Deoxyribose Nucleic Acid) identification and screening to obtain a four-transgenic mouse of which the genotype is SCL-tTA / Bcr-abl / LYZ2-CreERT2 / RUNX1. The invention provides a solid research basis for deep research of the effect of the RUNX1 gene in chronic myelogenous leukemia and research and development of LSCs targeted drugs aiming at the RUNX1 gene.

The invention relates to a detection method and a kit for detecting TP53 gene mutation based on dPCR, primers and probes of the detection method are designed according to a TP53 gene DNA sequence, a pair of primers and two Taqman probes are respectively designed aiming at 6th and 8th exon gene sequences of the TP53 gene, the sequences of the primers and the probes are the same as the sequence of a wild type TP53 gene, and the TP53 gene mutation can be detected by the detection method and the kit. The two probes on each exon are mutually referenced, and when mutation such as insertion, replacement and deletion exists in a probe coverage area, the corresponding probe cannot be combined with a template and is subjected to enzyme digestion to emit fluorescence, so that the probe can detect various mutation of the TP53 gene. The detection method comprises the following steps: extracting sample DNA, preparing a dPCR reaction mixed solution, preparing micro-droplets, carrying out PCR amplification, collecting signals and calculating the concentration. The kit for detecting TP53 gene mutation is easy to operate, high in sensitivity, low in price and suitable for continuous monitoring of mutation cloning loads of patients.

The application provides an HLA gene amplification primer based on a third-generation sequencing platform and an application thereof.The HLA gene amplification primer comprises a combination of any one or any multiple groups of nine groups of primers.The amplification primer designed for 11 target points of HLA not only realizes near-full-length amplification of the HLA gene, but also improves the specificity of amplification, and further improves the uniformity of HLA gene sequencing data.In addition, the amplification primer for 11 target points of the HLA gene in the application realizes one-pot detection in the same reaction system, and is convenient to operate.

The present invention relates to a Hepatocyte detection kit, characterized in that it comprises a capture probe for CPS1 gene mRNA and a signal amplification system. By designing the capture probe composition structure, the capture probe is designed as a dual-probe system and an HNA sequence modification is introduced, thereby ensuring high sensitivity, high specificity, high efficiency and high stability of capture probe hybridization, so that the capture probe in the kit of the present invention can be well applied to the probe detection system of the present invention, while improving the detection sensitivity, specificity and stability while shortening the detection time; by optimizing the introduction of multiple groups of P5 sequences at the 5' end and 3' end of the amplification probe in the signal amplification system and introducing PNA sequence modification into the labeling probe, the signal amplification system in the kit of the present invention has a more efficient signal amplification function, while improving the signal-to-noise ratio and accuracy of the detection, and further shortening the detection time.

Disclosed herein are nucleic acid compositions and methods of use. The nucleic acid compositions may have a therapeutic nucleic acid sequence operably linked to a nuclear targeting sequence that increases expression of the therapeutic nucleic acid in a cell by at least 1.25 fold; at least 80% sequence identity to SEQ ID NO: 6; or a first regulatory element comprising a promoter sequence operably linked to a hemoglobin subunit gamma intron (hBGi) sequence, and a second regulatory element comprising a woodchuck hepatitis posttranscriptional regulatory element (WPRE) sequence. The nucleic acid compositions with these features may enhance therapeutic nucleic acid transfection and expression. Also disclosed herein are transgenes optimized for gene therapy applications, including novel FVIII transgene sequences, in which the B domain may be non-naturally occurring the A1 and / or A3 domain may include at least one amino acid substitution.

The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the CIDEB gene, as well as methods of inhibiting expression of CIDEB, and methods of treating subjects that would benefit from reduction in expression of CIDEB, such as subjects having a CIDEB-associated disease, disorder, or condition, using such dsRNA compositions.

The invention discloses development and application of carbapenem drug-resistant gene detection test paper based on CRIPSR-dCas9. A primer pair, an sgRNA sequence and a recognition probe sequence aiming at an NDM gene are as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. The invention also discloses a preparation method of the carbapenem drug-resistant gene detection test paper based on CRIPSR-dCas9 and application of the carbapenem drug-resistant gene detection test paper based on CRIPSR-dCas9. According to the primer pair aiming at the KPC gene, an sgRNA sequence and an identification probe sequence are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8. The invention further discloses a kit for detecting the KPC gene. The method is simple to operate and high in specificity, extra expensive equipment assistance is not needed in the whole detection process, and specific and sensitive detection of common carbapenem drug-resistant genes such as NDM, KPC and OXA-48 in clinical samples can be achieved within 40 min.

The invention discloses a kit for detecting LINC01480 gene expression as well as a preparation method and application thereof, and relates to the field of gene expression kits. The kit for detecting the expression of the LINC01480 gene comprises a specific primer pair aiming at the LINC01480 gene, a specific primer pair aiming at a reference gene GAPDH, a positive control lentiviral vector containing a LINC01480 gene segment, a negative control lentiviral vector and an RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) reaction system. According to the present invention, the MRD of the treated MM patient is noninvasively monitored by detecting the LINC01480 gene expression so as to achieve the purpose of improving the convenience and the accuracy of the MRD index monitoring of the treated MM patient.

The invention discloses an NPM1 mutation dynamic monitoring method based on a microdroplet digital PCR technology, and relates to the technical field of molecular diagnos.The method comprises the steps that S1, RNA is extracted from a sample; s2, carrying out reverse transcription on the extracted RNA to obtain cDNA (complementary deoxyribonucleic acid); s3, designing a specific primer and a fluorescent probe aiming at the NPM1 gene exon 12 A type mutation, and designing a primer and a fluorescent probe of an ABL1 gene at the same time; s4, verifying the designed primer and probe and confirming the performance of a reaction system; s5, preparing a reaction mixed solution; s6, carrying out oil drop generation on the reaction mixed solution; specific primers and a fluorescent probe are designed for NPM1 gene exon 12 type A mutation, ABL1 is used as a reference gene, a droplet digital PCR platform is used for detection, the amplification efficiency and specificity of the primers and the probe are verified through real-time fluorescent quantitative PCR, the concentration combination of the primers and the probe is optimized by using a clinical sample, and the kit is used for detecting the NPM1 gene exon 12 type A mutation. And a plurality of samples are adopted for cross validation, so that high sensitivity and specificity of the method on NPM1 mutation detection are ensured.

The application provides a kit for detecting BRCA1 / 2 genes of preimplantation embryos and application thereof. The method is based on transcriptome detection, reduces the possibility of tripping, and improves the accuracy and success rate of BRCA1 / 2 gene detection in preimplantation embryo cells.