Controlling element for human esophagus cancer cell Fascin gene promoter region

A technology for gene promoter regions and regulatory elements, applied in genetic engineering, plant genetic improvement, biochemical equipment and methods, etc.

Inactive Publication Date: 2007-08-15
SHANTOU UNIV MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are few reports on the regulation mechanism

Method used

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  • Controlling element for human esophagus cancer cell Fascin gene promoter region
  • Controlling element for human esophagus cancer cell Fascin gene promoter region
  • Controlling element for human esophagus cancer cell Fascin gene promoter region

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Embodiment 1 Containing the construction of the eukaryotic expression plasmid of Fascin gene promoter

[0018] 1. Cloning and sequence analysis of the 5′ upstream fragment (-2900~+122) of human Fascin gene

[0019] According to the human Fascin gene sequence published in the literature, a pair of primers F2 and R were designed and synthesized to amplify the 3022 bp fragment upstream of the translation initiation codon of Fascin. The primer sequences are as follows:

[0020] F2, 5'-GTCGACCAGTCAAGACCTAGCACAGGGCTCAGG-3';

[0021] R,5'- AAGCTT GGTGGCAGTAGACGAGAGGCCGCTG-3' (Hind III site underlined). Using this pair of primers, a DNA fragment of about 3000 bp consistent with expectations was amplified from human esophageal cancer cell EC109 genome DNA by PCR. The amplified target fragment was recovered and connected to pMD18-T vector (TaKaRa Company), which contained Kpn I site. Then Kpn I / Hind III double enzyme digestion was performed to recover a DNA fragment of about ...

Embodiment 2

[0036] Example 2 Cell Culture, DNA Transfection and Fascin Gene Promoter Activity Analysis of Human Esophageal Cancer Cells

[0037] Use the QIAprep Spin Miniprep Kit to extract the experimental plasmid to be transfected, the control plasmid pGL3-Basic (Vector) and the internal reference plasmid pRL-TK, and determine their content and purity. Dilute the experimental plasmid and control plasmid expressing firefly luciferase to 100ng / μl with Buffer EB (10mM Tris Cl, pH8.5), and dilute the internal reference plasmid pRL-TK expressing Renilla luciferase to 100ng / μl with Buffer EB. 20ng / μl. The experimental plasmid and the internal reference plasmid were mixed at a ratio of 50:1, that is, 1 μg:20ng (10 μl:1 μl).

[0038] Esophageal cancer cells EC109 were routinely subcultured and inoculated in 96-well culture plates with 0.1ml per well. After 24 hours, the cells can be used for transfection when they reach 50%-60% confluence.

[0039] The transfection method is as follows: In t...

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PUM

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Abstract

The invention discloses a human cancer of the esophagus cell Fascin1 genetic promoter zone control element, which is characterized by the following: first judging human cancer of the esophagus cell Fascin1 genetic promoter; inserting gradually deleting promoter sequence of 5'end sequence into report gene advanced position; constructing a series of eucaryon expressing plasmid; expressing rotate-dying mammalian cell promptly; confirming key point controlling element -74- -41 of human Fascin1 genetic promoter transcripting active in cancer of the esophagus cell. This invention possesses important meaning for researching expression regulate and control mechanism of Fascin1 in tumor cell and clinical treatment with Fascin1 as target dot.

Description

technical field [0001] The invention relates to gene expression regulation, in particular to a regulatory element of Fascin gene promoter region of human esophageal cancer cells. Background technique [0002] Esophageal cancer is a common malignant tumor in my country, with a high incidence in some areas of Henan, Hebei, Shanxi, Jiangsu and Sichuan provinces. The incidence rate of esophageal cancer is also high in Chaoshan coastal area of ​​Guangdong Province, which is one of the six high-incidence areas of esophageal cancer in China and the only coastal area with high incidence of esophageal cancer. At present, the pathogenesis of esophageal cancer is still unclear. Our recent research results show that the upregulation of Fascin expression is an early event in the occurrence of esophageal cancer, and its overexpression not only promotes the division and proliferation of esophageal cancer cells, but also is closely related to the invasion, migration and adhesion of cancer ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C12N15/85
Inventor 许丽艳郭张燕高书颖杨章民谢剑君李恩民
Owner SHANTOU UNIV MEDICAL COLLEGE
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