Method for transforming marrow cells to establish cell line by mouse leukemia viruses and application thereof
A mouse leukemia virus, mouse technology, applied in microorganism-based methods, biochemical equipment and methods, and cells modified by introducing foreign genetic material, etc., can solve the problem of cumbersome classical methods, low success rate, and difficult to infect cells. Formation and other problems, to achieve the effect of good cell growth and stable expression
Active Publication Date: 2013-04-03
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology
Although some scholars use the bicistronic retroviral vector system to study the mutual regulation of Abl and other genes, and there are also reports on the application of mouse leukemia virus to transform mouse cells, the classic method of using mouse leukemia virus to transform cells is very cumbersome and difficult. Low success rate, and it is difficult for infected cells to form immortalized cell lines that can be passaged permanently
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Embodiment 1
[0035] Example 1. Establishment of an Immortalized Mouse Lymphocyte Line Expressing the Oncogene of Abelson Murine Leukemia Virus
[0036] 1. Acquisition of Abelson Murine Leukemia Virus (A-MuLV)
[0037] 1. Packaging of A-MuLV
[0038] (1) Construction of plasmid pLNCX-v-Abl carrying the oncogene v-Abl of Abelson murine leukemia virus
[0039] Primers were designed according to the nucleotide sequence of the v-Abl gene reported by Genbank (AF033812), and both upstream and downstream primers had HindIII restriction sites. The sequence specificity was confirmed by BLAST, and it was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The v-Abl gene shown in Sequence 1 in the sequence table was amplified by PCR, treated with HindIII digestion, connected with the pLNCX vector (Clontech, Cat.No.631503) plasmid that had been digested with HindIII, and transformed into Escherichia coli competent cells, Screen positive clones, extract plasmids, and send them ...
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The invention discloses a method for transforming marrow cells to establish a cell line by mouse leukemia viruses and application thereof. The construction method provided by the invention comprises the following step of: transforming invitro mouse marrow source lymphocyte by utilizing Ableson mouse leukemia viruses to obtain a mouse lymphocyte line for expressing the oncogene of the Ableson mouse leukemia viruses, wherein the oncogene of the Ableson mouse leukemia viruses is used for coding a protein represented by a sequence 2 in a sequence table. The mouse lymphocyte line for expressing the oncogene of the Ableson mouse leukemia viruses, which is constructed by the method provided by the invention, can passage for one hundred and thirty times and the cell has a good growing state and does not have the cell apoptotic phenomenon; and the expression condition of v-Abl protein is very stable so that an immortal cell line which can passage permanently is formed.
Description
technical field [0001] The invention relates to a method for establishing a mouse immortalized cell line and its application, in particular to a method for establishing a cell line by transforming bone marrow cells with mouse leukemia virus and its application. Background technique [0002] Abl is an important oncogene that can induce lymphoma / leukemia, including v-Abl, BCR-Abl, Tel-Abl and so on. v-Abl is an oncogene of Abelson murine leukemia virus (A-MuLV for short). Studies have shown that A-MuLV mainly induces malignant transformation of mouse lymphocytes, thereby triggering the generation of acute lymphoma in mice. BCR-Abl is caused by the fusion of BCR (breakpoint cluster region) and the C-terminus of c-Abl due to human chromosome translocation (chromosomal translocation), and is the most critical oncogene inducing human chronic myelogenous leukemia (CML) . Despite many years of research, the mechanism of Abl oncogene-induced cell carcinogenesis is still not fully ...
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IPC IPC(8): C12N15/86C12N5/10C12Q1/02G01N33/68C12R1/91
Inventor 陈吉龙杨建岭郭桂杰陈玉海
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI