Method for screening and identifying functional lncRNA

A construct and genome technology, applied in biochemical equipment and methods, other methods of inserting foreign genetic materials, recombinant DNA technology, etc., can solve problems such as the effectiveness of transcription knockdown

Active Publication Date: 2019-10-18
PEKING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although studies have demonstrated that based on RNA interference 10,11 or CRISPR 12 Screening is effective for identifying lncRNA functions, but RNAi approaches have potential off-target issues 13 , and both approaches are limited by the effectiveness of transcriptional knockdown

Method used

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  • Method for screening and identifying functional lncRNA
  • Method for screening and identifying functional lncRNA
  • Method for screening and identifying functional lncRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0142] Materials and methods

[0143] 1. Cells and Reagents

[0144] 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, CellMax BL102-02) and 1% penicillin / streptomycin at 37°C in 5% CO 2 cultivated in.

[0145] 2. Reverse transcription PCR (RT-PCR) to test for intron retention or exon skipping

[0146] 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 hour...

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Abstract

The invention relates to a method for performing gene interference on non-coding RNA (lncRNA) through targeting eukaryocyte genome splicing sites.

Description

[0001] field of invention [0002] The present invention relates to performing gene interference on long non-coding RNA (lncRNA) by targeting splicing sites in eukaryotic cell genome, thereby screening and identifying functional lncRNA. [0003] Background of the invention [0004] As a powerful genome editing tool, the CRISPR-Cas9 system has been used to identify gene function through large-scale screening 1-4 . Even at the genome scale, gene interference is mostly achieved through frameshift mutations generated within exons. In addition to about 2% of protein-coding genes in the human genome, there is more evidence that the remaining large transcripts are non-coding RNAs 5 . Among them, lncRNAs >200 nucleotides represent the majority of genes with no apparent protein-coding potential 6-7 . Previous studies have shown that the total number of human lncRNAs exceeds the total number of protein-coding genes and the number continues to climb 8 . [0005] lncRNAs regulate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N9/22C40B40/06
CPCC12N9/22C12N15/902C40B40/06
Inventor 魏文胜刘莹曹中正王轶楠郭昱袁鹏飞
Owner PEKING UNIV
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