Reporter system for researching expression regulation of Kiss1 gene and construction method of reporter system

A reporter system and gene expression technology, applied in the field of molecular biology, can solve problems such as the lack of a reporter system for Kiss1 expression regulation

Active Publication Date: 2017-03-29
DONGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no reporter system that can be used to study the regulation of Kiss1 expression in vitro

Method used

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  • Reporter system for researching expression regulation of Kiss1 gene and construction method of reporter system
  • Reporter system for researching expression regulation of Kiss1 gene and construction method of reporter system
  • Reporter system for researching expression regulation of Kiss1 gene and construction method of reporter system

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Experimental program
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Effect test

Embodiment 1

[0044] 1. Construction of the red fluorescent protein targeting vector Ai9-dtomato, the flow chart is as follows figure 1 shown.

[0045] (1) Linearization of Ai9 plasmid

[0046] According to the instructions of the PacI restriction endonuclease (NEB company), the enzyme digestion system was designed to digest the Ai9 plasmid, such as figure 2 Shown in A is the enzyme digestion result detected by 1% agarose gel electrophoresis.

[0047] (2) Removal of the Stop terminator sequence on the linearized Ai9 and plasmid circularization

[0048] According to the instructions of Cre enzyme from NEB company, the enzyme digestion system was designed, and the Stop terminator sequence between the two loxp sites in the same direction was excised by using Cre recombinase. Then, T4 ligase was used to ligate overnight at 16°C to achieve circularization of the linearized Ai9 plasmid with the Stop termination sequence removed.

[0049] (3) Colony PCR screening of Ai9-dtomato vector

[00...

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PUM

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Abstract

The invention relates to a reporter system for researching expression regulation of a Kiss1 gene and a construction method of the reporter system. The system is a dual-fluorescence Kiss1 gene expression regulation reporter system that red fluorescence protein achieves stable expression and green fluorescence protein is regulated by virtue of a Kiss1 expression regulatory element. The construction method comprises the following steps: constructing a red fluorescence protein targeting vector Ai9-dtomato; constructing a green fluorescence protein targeting system; constructing Cas9-GT1-7-Kiss1-2A-EGFP cells that EGFP is controlled by virtue of the Kiss1 expression regulatory element; and interpolating the cells into a red fluorescence background which is stable in expression, so as to obtain the reporter system. According to the reporter system and the construction method thereof provided by the invention, quantitative evaluation of a researched object on a Kiss1 action effect is achieved.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a reporter system and a construction method thereof for studying the regulation of Kiss1 gene expression. Background technique [0002] Since Lee et al. first discovered the Kiss1 gene with tumor metastasis suppressing effect in melanoma cells in 1996 (Lee JH, Welch DR (1997). Identification of highly expressed genes inmetastasis-suppressed chromosome 6 / human malignant melanoma hybrid cells using subtractive hybridization and differential display. Int J Cancer.; 71:1035-44.), after 20 years of research, people's understanding of the function of the Kiss1 gene has become increasingly clear. Existing studies have shown that Kiss1 is involved in the regulation of various life processes, such as sexual development (Gottsch, M.L., Cunningham, M.J., Smith, J.T. et al (2004). A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology 145(...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/65
Inventor 周宇荀李晓宁王斯佳李文文肖君华李凯
Owner DONGHUA UNIV
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