Method for constructing neural tube defect cell models and cell bank thereof by introducing SV40LT and hTERT recombination genes

A technology for recombining genes and cell models, applied to cells modified by introducing foreign genetic material, recombinant DNA technology, libraries, 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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  • Abstract
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  • Claims
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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 immortalized cell models and cell banks to study the pathogenesis of neural tube defects, and it is impossible to carry out research on related projects

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  • Method for constructing neural tube defect cell models and cell bank thereof by introducing SV40LT and hTERT recombination 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 Restriction digestion of 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. 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 0.5ul each of restriction endonuclease EcoR I ...

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Abstract

The invention relates to a method for constructing neural tube defect cell models and a cell bank thereof by introducing SV40LT and hTERT recombination genes in the field of birth defect intervention. The method is mainly characterized by comprising the following steps: connecting the product of BamHI enzyme digestion plasmids SV40LTag DNA and a vector pcDNA3.1(-)DNA by T4DNA to construct an SV40LTag-pcDNA3.1(-) recon; connecting the product of EcoR I and Xho I double enzyme digestion plasmids pCIneo-hTERT and a vector pLXSNneo by Ligation Mix to construct a pLXSNneo-hTERT recon, transfecting the two recons in neural tube defect cells through competent cell amplification, purification and authentication, screening the cells with the positive recon through G418 for cloning and carrying out enlarging cultivation, and screening the cells which meet the characteristics of immortalized cells and are the same as or similar to primary cells as the neural tube defect in-vitro study cell models mediated by the SV40LT and hTERT recombination genes, for laying the foundation for long-term in-vitro study for the pathogenesis of the cell models from a cell level.

Description

technical field [0001] The present invention relates to the method of introducing SV40LT (simian kidney virus 40 large T antigen gene) and hTERT (telomerase reverse transcriptase catalytic subunit) recombinant gene (mediated by SV40LT and hTERT) to construct neural tube defect cell model and cell bank, It is mainly used in the field of birth defect intervention research, providing cell models for in vitro research on neural tube defects and preserving its scientific research resources. Background technique [0002] Neural tube defects, also known as neural tube defects, are common and serious birth defects. The neural tube is the central nervous system of the fetus. From the 15th to the 17th day of the embryo, the nervous system begins to develop. Around the 22nd day of the embryo, the two sides of the neural folds begin to approach each other, forming a tube called the neural tube. The front end of the neural tube is called the anterior hole of the neural tube, and the tai...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C40B50/06C40B40/02
Inventor 翁炳焕罗玉琴孙义锡李晓杨燕梅黄荷凤
Owner 翁炳焕
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