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Process for effcient raising gene fixed-point conversion (mutating/repairing)

A gene and target site technology, applied in the field of improving intracellular gene-directed conversion, can solve problems such as simple understanding, and achieve the effect of improving efficiency

Inactive Publication Date: 2005-06-08
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI +1
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Problems solved by technology

[0008] The premise of effectively improving efficiency is to clarify the mechanism of action of this process. Unfortunately, people's understanding of the mechanism is still very shallow

Method used

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  • Process for effcient raising gene fixed-point conversion (mutating/repairing)
  • Process for effcient raising gene fixed-point conversion (mutating/repairing)
  • Process for effcient raising gene fixed-point conversion (mutating/repairing)

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Embodiment

[0094] In this example, in order to optimize the research system, the inventor constructed a mammalian cell system, which was deposited in the General Microorganism Center of China Committee for the Collection of Microorganisms on November 13, 2003, and its preservation number is CGMCC1030. The name is HeLa-mEGFP-F5. The following experiments are research work carried out in this system, the results of which illustrate the invention, but do not limit the scope of the invention.

[0095] Site-directed repair of the mutated green fluorescent protein EGFP gene

[0096] 1. Establishment of mammalian cell fluorescence reporter system

[0097] In order to obtain a stable, sensitive and easy-to-detect research system, the inventor's research team first designed a mammalian cell fluorescence reporter system.

[0098] Use artificial PCR method to mutate plasmid pEGFP-C1 in vitro (commercialized plasmid, purchased from ClONTECH company), design primers according to the sequence of pla...

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Abstract

A method for increasing the efficiency of site-specific gene conversion (mutation / reparation) features use of thymidine as the paralyser of DNA synthesis to decrease the moving speed of replicating fork, so artificially creating to condition for the conversion at target site and increasing the conversition efficiency by 10 times. A method for configuring a report system for the site-specific gene conversion and a cell line obtained by the method are also disclosed.

Description

technical field [0001] The present invention relates to the field of genetic engineering. Specifically, the present invention relates to a method for improving gene-directed conversion in cells. The present invention also relates to a cell line with the preservation number CGMCC1030 and a method for constructing the cell line. Background technique [0002] With more and more extensive knowledge of biological genome information, inventors can purposefully change and modify the genetic material of organisms, so as to obtain the results expected by the inventors. For example, inventors can prepare attenuated and inactivated vaccines by modifying the genetic material of viruses, and change the genetic material of plants and crops to obtain improved varieties. For genetic diseases caused by some congenital point mutations in humans and animals, it can play a role in genetic effect of treatment. In addition, this technology is also an important technical method in the current e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/10C12N15/09C12N15/82C12N15/90
Inventor 刘德培吴雪松辛立殷文璇黄建东谢赏恩梁植权
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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