Construction of neural tube defect cell model and cell bank thereof under induction of hTERT recombinant

A cell model, recombinant technology, applied in recombinant DNA technology, cells modified by introducing foreign genetic material, libraries, etc., can solve problems such as inability to carry out research, and achieve the effect of normal differentiation and maintenance of chromosomes

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

[0009] However, there have been no literature reports on the construction of immortal cell models and cell banks using hTERT to study the pathogenesis of neural tube defects from the cellular level in vitro, and it is impossible to carry out research on related projects

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  • Construction of neural tube defect cell model and cell bank thereof under induction of hTERT recombinant

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Embodiment Construction

[0015] 1. Extraction of hTERT: ① Digestion of pClneo-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. 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 restriction endonuclease EcoR I and Xho I 0.5ul each, incubate at 37°C for 1h, heat at 75°C for 15min, inactivate the enzyme, add 5uL electrophoresis sample buffer (also by adding 0.5mol / L EDTA) The reaction was terminated, and hTERT was amplified according to the conventional PCR method, and 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 gel makin...

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Abstract

The invention discloses construction of a neural tube defect cell model and a cell bank thereof under induction of an hTERT recombinant and relates to medical field. The construction is characterized in that incision enzymes EcoRI and XhoI are used for carrying out double-enzyme digestion on a plasmid pCIneo-hTERT and a carrier pLXSNneo, and Ligation Mix is used for connecting hTERT and pLXSNneo enzyme-digested products subjected to PCR amplification and gel electrophoresis separation to construct a pLXSNneo-hTERT recombinant; DH5a competent cells are transformed to purify, amplify and extract plasmids; the neural tube defect cells in logarithmic growth during in-vitro passage are subjected to liposome transfection so that the recombinant and the cell DNA are integrated and subjected to enlarged cultivation; clones containing positive recombinants are subjected to G418 screening; the cells, of which cellular morphology, growth curves, karyotype, nude mouse tumorigenesis tests, cell telomerase activity, hTERT mRNA expression, immunohistochemistry, and cell generation cycles and apoptosis rate meet immortal cell characteristics, and which are the same or similar to primary cells, are screened to be used as the neural tube defect cell model constructed under induction of hTERT to be frozen in liquid nitrogen. Therefore, the foundation is laid for long-time in-vitro researches on pathogenesis of related diseases from cellular level.

Description

technical field [0001] The invention relates to hTERT (telomerase reverse transcriptase catalytic subunit) recombinants to induce (mediated) construct neural tube defect cell models and cell banks, which are mainly used in the field of human reproductive health research and provide for in vitro research on neural tube defects. Cell models and preserve their 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 tail is called the posterior hole of the neural tube. On the 24th-25th and 26th day of the embryo, the anterior ho...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C40B50/06C40B40/02
Inventor 翁炳焕舒静李晓沈国松杨燕梅金帆
Owner 翁炳焕
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