Construction of neural tube defect cell model and cell bank of neural tube defect cell by importing SV40T gene

A cell model and neural tube technology, which can be applied to cells, libraries, and chemical libraries modified by introducing foreign genetic material, which can solve problems such as inability to carry out research projects.

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

[0008] However, there have been no literature reports on the construction of immortal cell models and cell banks that can be used to study the pathogenesis of neural tube defects from the cellular level in vitro, and it is impossible to carry out related research projects

Method used

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  • Construction of neural tube defect cell model and cell bank of neural tube defect cell by importing SV40T gene

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Embodiment Construction

[0014] 1. Extraction of SV40 large T antigen DNA: ① SV40 DNA digestion: 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 and 18uL H 2 O, add 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 ) to stop the reaction for electrophoresis. ②SV40 DNA electrophoresis: Take electrophoresis grade agarose and use electrophoresis buffer to make 10% agarose gel, pour it on the sealed gel filling platform, insert the sample comb, and remove the sealing tape from the gel making platform after the gel is solidified , pull out the comb, put it into the electrophoresis tank with enough electrophoresis buffer, the buffer is about 1mm above the surface of the gel, prepare the DNA sample with an appropriate amount of 10× loading buf...

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Abstract

The invention relates to construction of a neural tube defect cell model and a cell bank of neural tube defect cell by importing an SV40T gene. The construction is mainly characterized by comprising the following steps: constructing an SV40LTag-pcDNA3.1(I) recon by T4DNA ligase, BamHI, pcDNA3.1(I)DNA and V40LTag DNA; purifying the recon by competent state escherichia coli; importing the recon into in-vitro passaged or logarithmically grown neural tube defect tissue cell digested by collagenase II through a lipofection transfection method to ensure that the recon is integrated with the NDA of the cell, carrying out enlarge cultivation on the G418 screened cell containing a positive recon to clone, and screening a cell of which the cellular morphology, growth curve, karyotype, soft agar colony growth, nude mouse tumorigenesis experiment, SV40 large T gene detection in transfection cell DNA, mRNA expression product measurement and DNA sequence measurement result accord with the immortalized cell characteristics and are same or similar to those of a primary cell as the SV40T mediated neural tube defect in-vitro study cell model which is cryopreserved in liquid nitrogen, so as to lay a foundation for the long-term research of the pathogenesis in vitro from the cell level.

Description

technical field [0001] The invention relates to the introduction of SV40T gene (simian kidney virus 40 large T antigen gene or SV40LT antigen gene) to construct a neural tube defect cell model and its cell bank, which is mainly used in the field of neonatal birth defect intervention research, and provides cells for the study of neural tube defects model and save its 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 an...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C40B50/06C40B40/02
Inventor 翁炳焕黄荷凤徐晨明李晓张雪琴陈雁
Owner 翁炳焕
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