41 results about "HTERT Gene" patented technology

The invention relates to a method for quantitatively detecting recombinant lentivirus titers. The method includes: infecting target cells with samples to be detected; extracting a genome DNA (deoxyribonucleic acid) in the infected target cells; then detecting the copy number of WPRE elements in the genome DNA and the copy number of hTERT genes; calculating the number of particles in recombinant lentivirus in the average target cells according to the copy number of the WPRE elements and the copy number of the tTERT genes; calculating the number of the particles of the recombinant lentivirus contained in the samples to be detected in unit volume according to the sum of the target cells and the number of the particles of the recombinant lentivirus in the average target cells to obtain the titers of the recombinant lentivirus in the samples to be detected. The method is simple in operation, and the recombinant lentivirus titers can be detected accurately under the condition of not using fluorescent markers.

The invention discloses an immortalized piglet oral mucosa epithelial cell line and an establishment method and application thereof. The establishment method comprises the following steps: carrying out primary culture of piglet oral mucosa epithelial cells; extracting and purifying eukaryotic expression plasmids pCI-neo-hTERT containing hTERT genes; replacing the culture medium of the cells 24 hours after the cells are transfected with the eukaryotic expression plasmids pCI-neo-hTERT; after 48 hours, adding a DMEM / F12 culture medium containing 500 mu g / ml of G418 for screening; marking positive clones under a high power lens, and transferring into a culture plate for culture; carrying out enlarged culture; screening for 1 month and not adding G418 into the culture medium any more, so that the cells obtained after culturing for over 50 generations are the immortalized cell line. The immortalized piglet oral mucosa epithelial primary cells are consistent and uniform in morphology.

A reversibly immortalized human pancreatic islet cell line containing an hTERT gene and an SV40T gene each interposed between a pair of LoxP sequences, characterized in that it is capable of producing insulin and enhancing expression of insulin after excising the hTERT gene and the SV40T gene, in particular, NAKT-13 (deposited with International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology, address: AIST Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan, deposited date: Sep. 4, 2003, accession number: FERM BP-08461) or a passage cell line thereof; a human pancreatic islet cell obtained by excising the hTERT gene and the SV40T gene from the reversibly immortalized human pancreatic islet cell line or passage cell line thereof; and use of these cells. By using the reversibly immortalized human pancreatic islet cell line of the invention insulin-producing cells can be easily and surely obtained in a number enough to meet the demand.

The invention provides a kit for detecting activity degree of telomerase in a whole blood or serum sample, belonging to the field of molecular biology detection. The kit comprises the following primers: (1) primer sequences of telomerase hTERT genes: hTERT-F: 5'-GGCCGATTGTGAACATGGA-3'; hTERT-R: 5'-CCTCTTTTCTCTGCGGAACGT-3'; and (2) probe sequences of telomerase hTERT genes: hTERT-P: 5'-TACGTCGTGGGAGCCA-3', the 5' end is connected with fluorescence reporter genes, and the 3' end is connected with fluorescence quenching genes. The kit for detecting activity degree of telomerase in the whole blood or serum sample has the advantages of high detection sensitivity, short analysis time and high stability.

The invention discloses an application of cisplatin combined with phTERTp-HRP / IAA in drugs for the treatment of cervical cancer cells, in particular to the strengthening role of the cisplatin on the activity of a hTERT gene promoter. The cisplatin and the phTERTp-HRP / IAA are commonly used in the synergy for killing the human cervical cancer cells, and the action mechanism thereof is further explored. The application adopts Hela cells for carrying out the study and evaluates the impacts of the cisplatin on the activity of the hTERT promoter through the expression of downstream luciferase reporter gene regulated by the hTERT promoter. The synergic killing role and the mechanism of the cisplatin and the hTERT promoter during the combined use for regulating the gene therapy of an HRP / IAA system are evaluated through the detection of cell proliferation rate, cell apoptosis rate and cell cycle. The results confirm that the cisplatin can strengthen the activity of the hTERT gene promoter; and when in combined use with the phTERTp-HRP / IAA, the killing role of the human cervical cancer cells is significant.

The invention relates to construction of a down's syndrome cell model and a cell bank of a down's syndrome cell by employing hTERT gene recombination applied to the field of medicines. The construction is mainly characterized by comprising the following processes: performing double-enzyme digestion on a plasmid pCIneo-hTERT and a carrier pLXSNneo by incision enzyme EcoR I and Xho I, and connecting hTERT and pLXSNneo enzyme-digested products which are processed by PCR amplification and gel electrophoresis separation with Ligation Mix; constructing a pLXSNneo-hTERT recombinant, and converting a DH5a competent cell for purifying amplifying and choosing ampicillin-resistant colonies to extract plasmids; transfecting down's syndrome cells which are subjected to in vitro passage by lipidosome and are in logarithmic growth; integrating the recombinants with the cell DNA, carrying out enlarging circulation, and screening cells containing positive recombinants by G418; and screening the cell of which cell morphology, growth curve, chromosome karyotype, nude mouse carcinogenicity test, transfected cell telomerase activity, hTERT mRNA expression, immumohistochemistry, cell generation cycle and cell apoptosis rate accord with the cell characteristics of an immortalized cell and are the same as or similar to those of a primary cell as an hTERT-mediated down's syndrome in-vitro study cell model and cryopreserved in liquid nitrogen, thus a foundation is laid for in vitro long-term research of pathogenesis from the cellular level.

Disclosed is a novel substance capable of regulating the expression of a telomerase reverse transcriptase gene in a cell of a mammal. A gene capable of regulating the expression of hTERT, comprising a nucleotide sequence depicted in SEQ ID No: 1 or 2. The expression of a telomerase reverse transcriptase gene can be inhibited by inhibiting the expression of the gene. By utilizing this mechanism, the expression of a telomerase reverse transcriptase gene can be regulated.

A reversibly immortalized human pancreatic islet cell line containing an hTERT gene and an SV40T gene each interposed between a pair of LoxP sequences, characterized in that it is capable of producing insulin and enhancing expression of insulin after excising the hTERT gene and the SV40T gene, in particular, NAKT-13 (deposited with International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology, address: AIST Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan, deposited date: Sep. 4, 2003, accession number: FERM BP-08461) or a passage cell line thereof; a human pancreatic islet cell obtained by excising the hTERT gene and the SV40T gene from the reversibly immortalized human pancreatic islet cell line or passage cell line thereof; and use of these cells. By using the reversibly immortalized human pancreatic islet cell line of the invention insulin-producing cells can be easily and surely obtained in a number enough to meet the demand.

The present invention relates to a method for detecting c.-124 C>T and c.-146 C>T mutations in the Htert gene promoter. The method uses reaction compositions that comprise amplification primers and genotyping probes. Another aspect of this invention relates to the primers and probes used to implement the aforementioned method, with sequences, identified as SEQ ID no.1 to SEQ ID no.6, that displayhigh specificity for these mutations, and to the compositions containing these primers and probes. The present invention further relates to a kit comprising the above-mentioned compositions for detecting c.-124 C>T and c.-146 C>T mutations in the Htert gene promoter by carrying out the method according to the present invention. The method, gene sequences, compositions and kit of the present invention can be advantageously used for detecting trace amounts of c.-124 C>T and c.-146 C>T mutations in biological samples, due to their high sensitivity and specificity for such mutations. The present invention can thus be applied in early detection, identification, detection of recurrence or prediction and monitoring of diseases associated with this type of mutations, such as bladder carcinomas, thyroid carcinomas, squamous cell carcinomas, basal cell carcinomas, melanomas, gliomas and hepatocellular carcinomas, inter alia, and ultimately provide the basis for defining a suitable treatment. Thus, the present invention pertains to the technical fields of medicine, pharmaceutics, molecular biology, biochemistry, human genetics and the like.

Disclosed is a novel substance capable of regulating the expression of a telomerase reverse transcriptase gene in a cell of a mammal. A gene capable of regulating the expression of hTERT, comprising a nucleotide sequence depicted in SEQ ID No: 1 or 2. The expression of a telomerase reverse transcriptase gene can be inhibited by inhibiting the expression of the gene. By utilizing this mechanism, the expression of a telomerase reverse transcriptase gene can be regulated.

Reversibly immortalized human islet cell lines containing an hTERT gene and an SV40T gene each located between a pair of LoxP sequences, characterized by being capable of producing insulin and the expression of insulin being enhanced after removing the hTERT gene and the SV40T gene, in particular, NAKT-13 (having been deposited with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Address: Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, 305-8566 JAPAN, Deposition date: 04 September, 2003, Deposition No.: FERM BP-08461) or passage cell lines thereof; human islet cells obtained by removing the hTERT gene and the SV40T gene from the reversibly immortalized human islet cell lines or passage cell lines thereof as described above; and use of these cells. By using the reversibly immortalized human islet cell lines as described above, insulin-producing cells can be easily and surely obtained in a number enough to meet demand.

Disclosed is a novel substance capable of regulating the expression of a telomerase reverse transcriptase gene in a cell of a mammal A gene capable of regulating the expression of hTERT, comprising a nucleotide sequence depicted in SEQ ID No: 1 or 2. The expression of a telomerase reverse transcriptase gene can be inhibited by inhibiting the expression of the gene. By utilizing this mechanism, the expression of a telomerase reverse transcriptase gene can be regulated.

The invention discloses a method to transduce full material stem cell among marrow with human end particle enzyme gene, which comprises the following steps: enzyme-cutting pGRN145 plasmid with Hpa I and Not I restriction enzymes; getting hTERT cDNA fragment; connecting to pLXSN carrier with the same restriction enzymes; constructing pLXSN-hTERT retooling expressing carrier; using LipofectAMINE liposome transduction agent box; preparing pLXSN-hTERT retooling expressing carrier floated liquid through compatible eating property incasing line PA317; proceeding transduction for full material stem cell among marrow; constructing the stem cell with high effective expressing exogenesis hTERT gene; double-sieving with low density homomycin; getting the stem cell. This invention can reach the goal to increase life cycle of full cytoplasma stem cell among marrow.

The invention relates to an immortalized tree shrew small intestinal epithelial cell line and its construction method and application, belonging to the field of cell technology. The present invention activates the telomerase activity of tree shrew intestinal epithelial cells by using lentivirus-mediated hTERT gene transfection to obtain the cell line, and can rapidly and effectively establish immortalized tree shrew intestinal epithelial cells, and the established immortalized Tree shrew intestinal epithelial cells have successfully overcome replicative senescence and acquired immortality, and the immortalized tree shrew intestinal epithelial cells have maintained a phenotype very similar to that of primary tree shrew intestinal epithelial cells, maintaining cell contact inhibition and cell proliferation. Density is limited, and CPE can appear after infection with reovirus, so it can be used for research on virus infection and the like.

The present invention relates to an indolinone compound, which is 1-(4-chlorobenzyl)-3-(hydroxyimino)-4-(4-(trifluoromethyl)phenyl)indoline-2 ‑ketone, which can effectively inhibit the activity of hTERT gene promoter, semi-quantitative PCR experiments and Western blot experiments show that this compound can effectively inhibit the transcription and expression of hTERT, and more importantly, this compound can effectively reduce the length of telomeres in HeLa cells. MTT assay and plate clone assay showed that the compound could inhibit the viability and proliferation of HeLa cells. DAPI staining experiment and flow cytometry analysis showed that the compound could promote cell apoptosis. These results all indicate that the compound can target and inhibit the expression of hTERT and can be used as a new antitumor drug.