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Tags and methods for simultaneous visualization of gene dna, mrna and protein in living cells

A living cell and labeling technology, which is applied in the field of DNA, mRNA and protein labeling, can solve the problems of limited application, lack, and inability to clearly capture the quantitative analysis of nascent RNA, and achieve the effect of expanding the scope of application and accurate quantification

Active Publication Date: 2021-12-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This work visualizes both mature mRNA and nascent RNA, thus failing to clearly capture nascent RNA production and quantify the dynamic regulation of transcription
In addition, the DNA of the three components of the reporter system is ~20kb in total, which can only be used as an exogenous reporter gene, which limits the application of this technology to a certain extent
[0005] Therefore, the existing technology lacks a live cell imaging technology that can be widely used to realize the visualization of DNA, mRNA and protein of genes at the same time, and lacks the ability to better study the expression regulation of endogenous genes. and other research work provide a powerful gene visualization tool

Method used

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  • Tags and methods for simultaneous visualization of gene dna, mrna and protein in living cells
  • Tags and methods for simultaneous visualization of gene dna, mrna and protein in living cells
  • Tags and methods for simultaneous visualization of gene dna, mrna and protein in living cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] S1. Design and construct a blue fluorescent protein sequence containing introns:

[0068] The human HeLa cell genome was extracted, and the blue fluorescent protein sequence containing introns was obtained by nucleic acid synthesis, such as figure 1 as shown in (a);

[0069] S2. The screened TS1 capable of efficiently targeting and editing the nematode genome 12x Sequence with reduced repetitiveness of MS2V5 12x The sequence is synthesized by nucleic acid, and the TS1 repeat unit and the MS2V5 repeat unit are alternately connected to form a DNA / RNA dual visualization sequence, such as figure 1 as shown in (b);

[0070] S3. Construction of TriTag tags: Insert the dual visualization sequence into the intron region of the blue fluorescent protein sequence containing introns to obtain TriTag tags using the enzyme-cut ligation method, such as figure 1 As shown in (c), specifically the BstxI restriction enzyme site inserted into the intron sequence.

Embodiment 2

[0072] (1) Construct a visualized stable cell line, such as figure 2 As shown in (a):

[0073] (1.1) On the same day, HEK293T cells were cultured with PS-free medium and spread into a 12-well plate to form the first cell culture medium. The concentration of HEK293T cells added could make the cell density reach more than 80% the next day;

[0074] (1.2) The next day, carry out lentiviral packaging: use 75 μl of serum-reduced medium (Opti-medium) to premix each 750ng of dCas9-GFP 14xAfter virus expression plasmid and stdMCP-tdTomato virus expression plasmid, pCMV-dR8.91 plasmid of 705ng and PMD2.G plasmid of 87ng, then add 4.5μl Fugene transient transfection reagent (Promega) to form liposome, add dropwise to the In the supernatant of the first cell culture medium, HEK293T cells in the first cell culture medium were transiently transfected to prepare cells containing virus particles;

[0075] (1.3) After 12 hours of transient transfection in step (1.2), suck off the supernata...

Embodiment 3

[0100] (1) Construct the target endogenous gene (human histone H2B gene and nuclear membrane protein LMNA gene) visualization system in a stable cell line and transfect and sort:

[0101] (1.1) According to the protein structure prediction of the target endogenous gene product H2B histone and LMNA nuclear membrane protein, choose to insert the TriTag tag into the C-terminus of the human histone H2B gene, and insert the TriTag tag into the N of the human nuclear membrane protein LMNA gene end. The human HeLa cell genome was extracted, and the homology arm sequences (5' homology arm sequence and 3' homology arm sequence) on both sides of the H2B gene C-terminus or LMNA gene N-terminus were respectively amplified, and the length of the homology arm sequence was about 500 bp. The HDR repair template of 5' homology arm sequence-TriTag tag-3' homology arm sequence was constructed by homologous recombination. Such as image 3 (a) shown.

[0102] (1.2) As in steps (2.2)-(2.4) in Ex...

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Abstract

The invention discloses a triple label and method for simultaneously visualizing DNA, mRNA and protein of genes in living cells. The tag contains a CRISPR-Tag DNA sequence composed of 12 TS1 repeat units, an MS2 DNA sequence composed of 12 MS2V5 unit sequences, and a blue fluorescent protein sequence containing introns. The DNA sequence is short in length and can be inserted into the N-terminal or C-terminal of the target protein-coding gene through CRISPR-Cas9-mediated gene recombination; the preparation method first prepares the TriTag tag, constructs a visualized stable cell line, and then constructs the target endogenous gene visualization system and Transfection and sorting, and finally live cell dynamic imaging can be realized. The TriTag of the present invention can be marked to realize dynamic imaging of chromatin, and can intuitively visualize the chromatin dynamics and gene expression of endogenous specific target genes, which is conducive to data integration and analysis, and helps to analyze the dynamic regulation mechanism of gene expression .

Description

technical field [0001] The invention relates to a method for establishing a TriTag labeling imaging system in the field of biotechnology, and its specific content relates to a label and method for simultaneously visualizing DNA, mRNA and protein of genes in living cells, so as to realize real-time imaging and analysis of gene expression regulation. Background technique [0002] Gene expression is the process of transferring genetic information from gene to protein. As two key steps of gene expression, transcription and translation are closely regulated throughout the process. However, few studies have visualized the entire process of gene expression regulation: that is, in living cells, the DNA, mRNA and protein of genes are visualized at the same time, revealing how genes regulate transcription at the chromatin level, and then realize the spatiotemporal regulation of protein expression. [0003] There has been a lot of work to visualize RNA in living cells. In these works,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/113C12N15/90C12N15/867C12N15/66C12N15/65C12N9/22C12N5/10
CPCC12N9/22C12N15/113C12N15/65C12N15/66C12N15/86C12N15/907C12N2740/15043C12N2310/20
Inventor 陈宝惠徐海月邹炜
Owner ZHEJIANG UNIV
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