Construction of trisomy 18 syndrome cell model and cell bank thereof by hTERT transgenosis

A trisomy, cell model technology, applied in the field of neonatal birth defect intervention research, can solve problems such as inability to carry out research, and achieve the effect of maintaining chromosomes

Inactive Publication Date: 2015-03-18
翁炳焕
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  • Description
  • Claims
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Problems solved by technology

[0010] However, there have been no literature reports on the construction of immortal cell models and cell banks using hTERT to study the pathogenesis of 18-trisomy at the cellular level in vitro, and it is impossible to carry out research on related projects

Method used

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  • Construction of trisomy 18 syndrome cell model and cell bank thereof by hTERT transgenosis

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Embodiment Construction

[0016] 1. Extraction of hTERT: ① Digestion of pClneo-hTERT: hTERT is located between the EcoRI and SalI sites of the plasmid pClneo-hTERT, and the multiple cloning site (MCS) of the pLXSNneo vector contains EcoRI and XhoI restriction sites. Commercially purchased pCIneo-hTERT plasmid, dissolved in an appropriate amount of ultra-clean H 2 In O or TE buffer, add 2uL 10× digestion buffer and 18uL H 2 O, add 0.5ul each of restriction endonuclease EcoR I and Xho I, incubate at 37°C for 1h, heat at 75°C for 15min, inactivate the enzyme, add 5uL electrophoresis loading buffer (also can be terminated by adding 0.5mol / LEDTA) For the reaction, after amplifying hTERT according to the conventional PCR method, the amplified product was collected for electrophoresis. ②hTERT electrophoresis: Take electrophoresis grade agarose and use electrophoresis buffer to make 10% agarose gel, pour it into the sealed gel filling platform, insert the sample comb, and remove the sealing tape from the gel ...

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Abstract

The invention relates to construction of an hTERT mediated trisomy 18 syndrome cell model and a cell bank thereof in the medical field. The construction is mainly characterized in that incision enzymes EcoR I and Xho I are used for carrying out double enzyme digestion on plasmid pCIneo-hTERT and carrier pLXSNneo, and Ligation Mix is used for connecting hTERT and pLXSNneo enzyme-digested products subjected to PCR amplification and gel electrophoresis separation to construct a pLXSNneo-hTERT recon; DH5a competent cells are transformed to purify, amplify and pick ampicillin-resistant bacterial colony extraction plasmids; trisomy 18 syndrome cells in logarithmic growth during in-vitro passage are subjected to lipofection transfection; the recons are integrated with cell DNAs and are subjected to amplification culture; positive recon colons are subjected to G418 screening; cells, which meet the immortalized cell characters in the aspects of cellular morphology, growth curve, karyotype, nude mice tumorigenesis test, transfected cell telomerase activity, hTERT mRNA expression, immunohistochemistry, and cell generation cycle and apoptosis rate and are as same as or similar to primary cells, are screened to be as the hTERT mediated trisomy 18 syndrome external research cell models to be frozen in liquid nitrogen. Therefore, the foundation of in vitro long-term research of pathogenesis of trisomy 18 syndrome based on the cellular level is established.

Description

technical field [0001] The invention relates to hTERT (telomerase reverse transcriptase catalytic subunit) transgene (mediated) construction of a 18-trisomy cell model and its cell bank, mainly used in the field of neonatal birth defect intervention research, which is 18-trisomy In vitro studies of syndromes provide cell models and preserve their research resources. Background technique [0002] Trisomy 18 is one of the major birth defects, also known as Edward's syndrome, that is, the patient has an extra chromosome 18. The main symptoms of this syndrome are kyphosis, narrow eye cleft, and ear Deformity, low ear position, small jaw, short sternum, etc. Due to severe mental retardation and multiple deformities in children, 50% died within 2 months after birth, less than 10% lived to 1 year, and very few could live to 15 years old. Due to the lack of effective treatment methods, once a child is born, it will bring a great mental and economic burden to the family and society...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C40B50/06C40B40/02
Inventor 翁炳焕李晓李红阁叶光勇郑金川金帆
Owner 翁炳焕
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