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Construction of trisomy 21 syndrome cell model and cell bank thereof by using recombined SV40LT and hTERT genes

A technology of trisomy syndrome and cell model, applied in combinatorial chemistry, recombinant DNA technology, cells modified by introducing foreign genetic material, etc., can solve problems such as inability to carry out research, and achieve the effect of long life

Inactive Publication Date: 2015-03-18
翁炳焕
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  • Claims
  • Application Information

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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 an immortal cell line, 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 trisomy 21, and it is impossible to carry out research on related projects

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  • Construction of trisomy 21 syndrome cell model and cell bank thereof by using recombined SV40LT and hTERT genes

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Embodiment Construction

[0015] 1. Extraction of SV40LT and hTERT: (1) Enzyme digestion of SV40LT and hTERT: ① Enzyme digestion of SV40LT: buy SV40 freeze-dried powder or SV40 plasmid containing large T antigen gene from the market, dissolve in appropriate amount of H 2 In O or TE buffer, add 2uL 10× digestion buffer, 18uL H 2 O and restriction endonuclease BamH I (1-5U / ugDNA), incubate at 37°C for 1h, heat at 75°C for 15min, inactivate the enzyme, add 5uL electrophoresis loading buffer (also by adding 0.5mol / L EDTA) Stop the reaction and prepare for electrophoresis. ② Digestion of 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. Purchase the pCIneo-hTERT plasmid from the market and dissolve it in an appropriate amount of ultra-clean H 2 In O or TE buffer, add 2uL 10× digestion buffer and 18uL H 2O, add restriction endonuclease EcoR I and Xho I 0.5ul each, incub...

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Abstract

The invention relates to construction of a trisomy 21 syndrome cell model and a cell bank thereof by using recombined SV40LT and hTERT genes in the reproduction and genetics fields. The construction is mainly characterized in that T4DNA is used for connecting products obtained by carrying out digestion on plasmid SV40LTag DNA and a carrier pcDNA3.1(-) DNA with a BamHi enzyme to construct an SV40LTag-pcDNA3.1(-) recon; Ligation Mix is used for connecting products obtained by carrying out double digesion on plasmid pCIneo-hTERT and a carrier pLXSNneo with EcoR I and Xho I to construct a pLXSNneo-hTERT recon; the two recons are subjected to competent cell purification and identification and then are transfected into trisomy 21 syndrome cells, positive cells are subjected to G418 screening and amplification culture, and then cells, which meet the immortalized cell characters and are as same as or similar to primary cells, are selected as an SV40LT and hTERT combined mediated trisomy 21 syndrome cell model. Therefore, the foundation of in vitro long-term research of pathogenesis of trisomy 21 syndrome based on the cellular level is established.

Description

technical field [0001] The invention relates to the construction of a 21-trisomy syndrome cell model and its cell bank by recombinant SV40LT (simian kidney virus 40 large T antigen gene) and hTERT (telomerase reverse transcriptase catalytic subunit) gene, mainly used in the field of reproductive genetic research , to provide cell models for in vitro research on trisomy 21 and preserve its scientific research resources. Background technique [0002] Trisomy 21, that is, an extra chromosome 21 in the patient's somatic cells, manifests as congenital mental retardation. There are 16,000-20,000 new cases of trisomy 21 syndrome in my country every year, which will cause economic losses of 7.3-9 billion yuan, causing serious psychological burden and mental pain to society and families, and has seriously affected the population quality of our country. become a heavy burden on social development and a serious obstacle to sustainable development. In recent years, Trisomy 21 has been ...

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C40B50/06C40B40/02
Inventor 翁炳焕
Owner 翁炳焕
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