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A method for constructing an efficient high-throughput promoter and enhancer element library

A promoter and enhancer capture technology, applied in the fields of genetic engineering, genetic engineering, and cell biotechnology, which can solve the problems of high-throughput capture of promoters and enhancers.

Inactive Publication Date: 2019-01-25
李其龙 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, high-throughput capture of promoters and enhancers is still a challenge

Method used

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  • A method for constructing an efficient high-throughput promoter and enhancer element library
  • A method for constructing an efficient high-throughput promoter and enhancer element library
  • A method for constructing an efficient high-throughput promoter and enhancer element library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] (1) Recovery and culture of 4T1 cells

[0079] a) Take out the cryopreserved mouse breast cancer cell line 4T1 from liquid nitrogen and put it directly in a water bath at 37°C, and take it out after 3-5 minutes for the cells to thaw; clean the cells with PRMI-1640 Suspend cells in cell culture medium, transfer to a 15mL centrifuge tube, and gently pipette to mix, centrifuge at 1500rpm for 5 minutes, pour off the supernatant; use 2mL of cell culture medium PRMI-1640, containing 10% fetal bovine serum and 1 % double antibody (ie 100μg / mL streptomycin and 100U / mL penicillin) to suspend the cells; put 8mL cell culture medium RPMI-1640 in the cell culture flask, containing 10% fetal bovine serum and 1% double antibody, Add 2 mL of cell suspension to the culture flask; then place the culture flask at 37°C, 5% CO 2 cultured in a cell culture incubator;

[0080] b) Adherent culture of mouse breast cancer 4T1 cells in RPMI-1640 cell culture medium containing 10% fetal bovine s...

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Abstract

The invention discloses a high-throughput promoter capture system, which inserts a genome random fragment into a BamHI / HindIII double-digested Pro- Free- Trap vector, and thus various transient transfection promoter screening systems Pro- DNA- Trapare obtained. Alkaline phosphatase reporter activity assays areperformed on potential promoter fragments (random genomic DNA), known promoter CMVs, andempty vectors by a microplate reader. If the recombinant plasmid expresses an alkaline phosphatase, it can react with the substrate p-nitrophenylphosphate disodium, and whether the potential promotersequence has promoter activity or not can be determined by detection.

Description

technical field [0001] The invention relates to the fields of cell biotechnology, genetic engineering and genetic engineering, and is a high-throughput capture promoter and enhancer method applicable to various transfection and transformation cells. It mainly involves connecting the plasmid (Pro-trap) into the random fragment of the genome, so as to construct a promoter screening recombinant plasmid Pro-promoter with random fragments. By detecting the expression intensity of the alkaline phosphatase reporter gene, it is known whether the random restriction fragment has promoter activity and activity intensity. Background technique [0002] Eukaryotic gene expression is regulated by various factors, such as transcription factors, miRNA, IncRNA, etc. At present, the regulation of gene expression can be divided into transcriptional regulation, translational regulation, and post-translational processing regulation. Among them, the regulation of transcription level is particula...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66
CPCC12N15/63C12N15/66
Inventor 马世良李其龙路瑶
Owner 李其龙