Construction of megakaryocyte model and cell bank of megakaryocyte by virtue of SV40 gene recombination

A megakaryocyte and gene recombination technology, applied in the field of medical blood disease research, can solve problems such as the inability to establish a megakaryocyte development megakaryocyte model

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

[0010] In order to solve the problem that it is currently impossible to establish a megakaryocyte model that can be subcultured in vitro for a long time and can be used to study megakaryocyte development, function, and pathological mechanisms in vitro, the inventors proposed the present invention

Method used

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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 0 or TE buffer, add 2uL 10× digestion buffer and 18uL H 2 0, 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 (or add 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 buffer, and th...

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Abstract

The invention relates to construction of an SV40LT gene-mediated megakaryocyte model and a cell bank of megakaryocyte, belonging to the field of medical research. The construction is mainly characterized in that an SV40LTag-pcDNA3.1 (-) recombinant is constructed from T4DNA ligase, BamHI, pcDNA3.1 (-) DNA and SV40LTag DNA by virtue of conventional means; the recombinant is purified by virtue of competent escherichia coli; the recombinant is introduced into megakaryocyte cultured in vitro through a lipofection transfection method, so that the recombinant is integrated with the DNA of the cell; a cell screened by virtue of G418 and containing a positive recombinant is subjected to subculture and enlarged cultivation; a cell which is consistent with the biological properties of an immortalized cell in cellular morphology, cell growth curve, karyotype, nude mice carcinogenicity test, detection of SV40 large T gene in transfection cell DNA, determination of an mRNA expression product and a DNA sequence determination result is screened out as a megakaryocyte model, and is cryopreserved in liquid nitrogen, so as to construct an SV40LT gene-mediated megakaryocyte model and the cell bank of megakaryocyte; therefore, a method for long-term in vitro subculture of the megakaryocyte is established, and the method is applicable to the in vitro research of development, function and pathogenic mechanism of the megakaryocyte.

Description

technical field [0001] The invention relates to SV40 gene (simian kidney virus 40 large T antigen gene or SV40LT gene) recombination (mediated) construction of a megakaryocyte model and its cell bank, which is mainly used in the field of medical blood disease research for the development of megakaryocytes or related to megakaryocytes Provides cell models for various research and preserves its scientific research resources. Background technique [0002] Megakaryocytes are mature cells in normal bone marrow that produce platelets. The cells are huge, and the edges of mature megakaryocytes rupture and fall off to form platelets. According to the development process, megakaryocytes are divided into (1) Primordial megakaryocytes: large cell body, 15-30 μm in diameter, round or irregular. The nuclei are large, round, and irregular. The chromatin is thick and reticular, closely arranged, and there are 2 to 3 nucleoli. Cell mass less, uneven, irregular edge, stained dark blue, no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C40B50/06C40B40/02
Inventor 翁炳焕黄荷凤蔡志坚潘玲李晓陈雁
Owner 翁炳焕
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