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High-signal-noise ratio negative heredity screening method

A construct and genome technology, applied in the field of negative genetic screening with high signal-to-noise ratio, can solve problems such as low signal-to-noise ratio

Inactive Publication Date: 2020-06-26
EDIGENE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current overall low signal-to-noise ratio for negative screening

Method used

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  • High-signal-noise ratio negative heredity screening method
  • High-signal-noise ratio negative heredity screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0113] Materials and methods

[0114] 1. Cells and Reagents

[0115] The HeLa cell line from Z. Jiang's laboratory (Peking University) was cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco C11995500BT). The Huh 7.5 cell line from the laboratory of S. Cohen (Stanford University School of Medicine) was cultured in DMEM (Gibco) supplemented with 1% MEM non-essential amino acids (NEAA, Gibco 1140-050). K562 cells from H. Wu laboratory (Peking University) and GM12878 cells from Coriell cell bank were cultured in RPMI1640 medium (Gibco 11875-093). All cells were supplemented with 10% fetal bovine serum (FBS, CellMaxBL102-02) and 1% penicillin / streptomycin at 37°C in 5% CO 2 cultivated in.

[0116] 2. Reverse transcription PCR (RT-PCR) to test for intron retention or exon skipping

[0117] Cloning of sgRNA into a lentiviral expression vector carrying a CMV promoter-driven mCherry marker, followed by transduction of HeLa by viral infection at MOIOC cell 1-4 , 72 hours...

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PUM

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Abstract

Through targetting splicing sites of eukaryocyte genomes, the signal-noise ratio through negative heredity screening can be increased.

Description

[0001] field of invention [0002] The present invention relates to carrying out gene destruction on messenger RNA (mRNA) of a gene by targeting the splicing site in the eukaryotic cell genome, so as to complete the negative screen of gene function more efficiently. [0003] Background of the invention [0004] An important purpose of genetic analysis is to identify genes responsible for specific biological phenotypes or diseases. Hypothesis or reverse genetic screens use known information to detect specific genetic variants through a "genotype-to-phenotype" research approach. Forward genetic screening is a powerful tool for discovering and annotating functional genetic elements. It modifies or regulates the expression of a large number of genes through the "phenotype-to-genotype" research method, and screens out cells or organisms that can produce special phenotypes. , and then analyze the mutations that lead to these phenotypes. [0005] Initially, forward genetic screening...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/79
CPCC12N15/79C12N15/902C12N2800/80C12N2810/10
Inventor 袁鹏飞王飞于玲玲董曦
Owner EDIGENE INC
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