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Building of klinefelter syndrome cell model and cell bank of klinefelter syndrome cell by virtue of recombinant SV40 gene

A cell model and syndrome technology, applied in the field of human reproductive health research, can solve problems such as inability to carry out research projects

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

[0007] However, there has been no literature report on the construction of an immortal cell model and its cell bank that can be used to study the pathogenesis of Klinefelter syndrome at the cellular level in vitro, and it is impossible to carry out related research projects

Method used

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  • Building of klinefelter syndrome cell model and cell bank of klinefelter syndrome cell by virtue of recombinant SV40 gene

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Embodiment Construction

[0013] 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 / LEDTA) Stop the reaction and prepare 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× load...

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Abstract

The invention relates to building of an SV40LT antigen gene-mediated klinefelter syndrome cell model and a cell bank of the klinefelter syndrome cell model, applied to the field of medical research. The building method is mainly characterized by comprising the following processes: building an SV40LTag-pcDNA3.1(1) recombinant by using T4DNA ligase, BamHI, pcDNA3.1(1) DNA and SV40LTag DNA according to a conventional method; purifying the recombinant by using competent escherichia coli, introducing the recombinant into an in vitro passage cell which is digested by collagenase II, or klinefelter syndrome histocyte which is in logarithmic growth, and integrating the recombinant with DNA; carrying out enlarging cultivation, screening cells containing positive recombinants by G418, and cloning; and screening a cell of which the cell morphology, the growth curve, karyotype, soft agar colony growth, nude mice tumor experiment result and transfection cell SV40 big T gene detection result, mRNA product measuring result and DNA sequencing result are in fit with the characteristics of an immortalized cell and are the same as or similar to that of a primary cell to be taken as an in vitro research cell model for the SV40LT-mediated klinefelter syndrome; and a foundation is laid for in vitro long-term research of pathogenesis of the klinefelter syndrome from the cellular level.

Description

technical field [0001] The invention relates to the construction of SV40 gene (simian kidney virus 40 large T antigen gene or SV40LT antigen gene) recombinant (mediated) Klinefelter syndrome cell model and its cell bank, which is mainly used in the field of human reproductive health research. It provides cell models and preserves its scientific research resources. Background technique [0002] Klinefelter syndrome is one of the chromosomal birth defects, also known as primary microorchidism or seminiferous tubule dysplasia, and is a relatively common male sex chromosome abnormality. The disease is characterized by an abnormal number of chromosomes, an extra X chromosome. The most common karyotype is 47,XXY. Some patients have more than two Xs, which are called variants of Klinefelter, such as: 48, XXXY, 49, xXXXY. Generally, the more Xs, the more severe the clinical symptoms. The incidence rate of this disease is 0.1%. Before puberty, there are generally no obvious sympt...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C40B50/06C40B40/02
Inventor 翁炳焕黄荷凤吕时铭朱瑞建王爱飞刘蓓
Owner 翁炳焕
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