Construction of down's syndrome cell model and cell bank of down's syndrome cell by employing hTERT gene recombination

Abstract

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.

Description

technical field [0001] The present invention relates to gene recombination (mediated) of telomerase reverse transcriptase catalytic subunit (hTERT) to construct a Down syndrome cell model and its cell bank, which is mainly used in the field of medical genetics research, and is an in vitro study of Down syndrome Provide cell models and preserve their research resources. Background technique [0002] Down syndrome, also known as trisomy 21, means that the patient has an extra chromosome 21, which is manifested as congenital stupidity. In my country, 16,000 to 20,000 cases of Down syndrome (trisomy 21 or congenital stupidity) are newly diagnosed every year, which will cause economic losses of 7.3 billion to 9 billion, and cause serious psychological burden and mental pain to society and families. , has seriously affected the population quality of our country, and has become a heavy burden on social development and a serious obstacle to sustainable development. [0003] In rece...

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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 Down syndrome from the cellular level in vitro, and it is impossible to carry out research on related projects

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