Establishment of cri du chat syndrome cell model and cell bank of cri du chat syndrome cell model through hTERT and SV40LT combined mediation

Abstract

The invention relates to a method for establishing a cri du chat syndrome cell model and a cell bank of cri du chat syndrome cell model through hTERT and SV40LT combined mediation, belonging to the field of reproductive health research. The method is mainly characterized by comprising the following steps: connecting T4DNA with a product obtained through digesting plasmid SV40LTag DNA and a vector pcDNA3.1(-) DNA by using BamHI enzyme, thereby establishing a SV40LTag-pcDNA3.1(-) recombinant; connecting Ligation Mix with a product obtained through double-enzyme digestion carried out on plasmid pCIneo-hTERT and a vector pLXSNneo by using EcoR I and XhoI, thereby establishing a pLXSNneo-hTERT recombinant, performing competent cell proliferation, purification and identification on the two recombinants, transfecting the tissue cells by using lipidosome, screening cells containing positive recombinants by using G418, cloning, carrying out amplifying culture, screening cells of which the cellular morphology, the growth curve, the karyotype, the inoculated nude mice experiment result, the transfection cell telomerase activity, the hTERT mRNA expression product, the immunohistochemical dyeing, the cell generation cycle and the cell apoptosis rate meet the characteristics of immortalized cells and are identical or similar to those of primary cells, as an in-vitro study cell model of the hTERT and SV40LT combined mediation cri du chat syndrome, and freezing and storing the cell model in liquid nitrogen. A foundation is laid for studying the pathogenesis in vitro for a long time at the level of cells.

Description

technical field [0001] The invention relates to hTERT (catalytic subunit of telomerase reverse transcriptase) and SV40LT (simian nephrovirus 40 large T antigen gene) to jointly mediate the construction of meowing syndrome cell model and its cell bank, mainly used in the field of reproductive health research, Provide cell models and preserve scientific research resources for the in vitro study of chromosomal deletion meowing syndrome. 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...

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Problems solved by technology

[0009] However, so far, there has been no information about simultaneously introducing the simian nephrovirus 40 large T antigen gene (SV40LT) and the catalytic subunit of telomerase reverse transcriptase (hTERT) into cells to establish immortal cell lines, and this construction can be used in vitro from the cellular level. Literature reports on the immortal cell model and its cell bank to study the pathogenesis of chromosomal deletion meowing syndrome, and it is impossible to carry out research on related projects

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