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Construction of chromosome deficiency cri du chat syndrome cell model and cell bank of chromosome deficiency cri du chat syndrome cell model by employing hTERT transfection

A chromosomal deletion and cell model technology, which is applied to cells, libraries, chemical libraries, etc. modified by introducing foreign genetic material, can solve the problems of inability to carry out research, lack of immortal cell model of cat meow syndrome and its cell bank, etc. The effect of preserving chromosomes

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

[0009] However, there has been no literature report on the establishment of an immortal cell model and its cell bank using hTERT to study the pathogenesis of chromosomal deletion meowing syndrome 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 chromosome deficiency cri du chat syndrome cell model and cell bank of chromosome deficiency cri du chat syndrome cell model by employing hTERT transfection

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Embodiment Construction

[0015] 1. Extraction of hTERT: ① Digestion of pClneo-hTERT: hTERT is located between the EcoRI and Sal I 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 of 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 ...

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Abstract

The invention relates to construction of a chromosome deficiency cri du chat syndrome cell model and a cell bank of the chromosome deficiency cri du chat syndrome cell model by employing hTERT transfection in the field of medicines. The construction is mainly characterized by comprising the following processes: connecting incision enzyme EcoR I and Xho I double-enzyme plasmid pCIneo-hTERT and a carrier pLXSNneo, and hTERT and pLXSNneo enzyme-digested products which are processed by PCR amplification and gel electrophoresis separation with Ligation Mix; constructing pLXSNneo-hTERT recombinants, and converting into DH5a competent cells to purify, amplify and extract plasmids; transfecting chromosome deficiency cri du chat syndrome cells which are subjected to in vitro passage and are in logarithmic growth by lipidosome; merging the recombinants with the cell DNA, carrying out enlarging circulation, and screening clones containing positive recombinants by G418; and screening cells of which cell morphologies, growth curves, karyotype, nude mice tumor experiment, cell telomerase activity, and hTERT mRNA expression, immunohistochemistry, cell generation cycle and apoptosis rate accord with immortalized cell characteristics and are the same as or similar to those of a primary cell to be used as an hTERT-transfected chromosome deficiency cri du chat syndrome cell model to be cryopreserved in liquid nitrogen, and a foundation is laid for long-term in vitro research on pathogenesis from cellular level.

Description

technical field [0001] The invention relates to hTERT (telomerase reverse transcriptase catalytic subunit) transfection (mediated) to construct a cell model of chromosomal deletion meowing syndrome and its cell bank, which is mainly used in the field of human reproductive genetic research, and is a chromosomal deletion meowing syndrome Provide cell models for in vitro studies of signs and preserve their scientific research resources. Background technique [0002] Chromosomal deletion cat call syndrome, also known as cri-du-chat syndrome, is a special disease first reported by French scientists in 1963. Cat-calling syndrome in children is caused by the deletion of the short arm of chromosome 5, and most of the deletions are the result of two breaks. Theoretically, there are at least four reasons for partial deletion of chromosomes: terminal deletion, middle deletion, translocation and short arm It was found that the missing part has 5p14, so 5p14 is considered to be the char...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C40B50/06C40B40/02
Inventor 翁炳焕舒静李晓罗玉琴沈国松黄荷凤
Owner 翁炳焕
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