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A method for terminating lncRNA biallelic transcription

A biallelic and gene technology, applied in the field of genetic engineering, can solve the problems of low incidence of homologous recombination, difficulty in gene knockout, etc., and achieve the effects of accurate gene function identification, improved accuracy, and accurate success rate.

Active Publication Date: 2019-11-05
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this technical solution is theoretically feasible, there may be many problems in actual operation, for example: 1) in mammalian embryonic cells, the incidence of homologous recombination is extremely low, therefore, if relying on the homologous recombination of the cell itself, Even if it can be achieved, allelic targeting is very difficult, and it is even more difficult to simultaneously target biallelic genes in the same cell; compared with traditional embryonic cell gene targeting, terminally differentiated cells are naturally The rate is low, which makes it more difficult to knock out genes relying on conventional homologous recombination; this is also the reason why the patent does not give examples of cell targeting, and the solution has no practical application value; 2) the technical solution does not An example of cell targeting is given. Even if the system uses homologous recombination, it can only be carried out according to traditional gene recombination. The homologous recombination arm of thousands of bp brings great difficulty to the construction of homologous recombination vector ; 3) The system only targets coding genes and does not involve the knockout of non-coding genes

Method used

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  • A method for terminating lncRNA biallelic transcription
  • A method for terminating lncRNA biallelic transcription
  • A method for terminating lncRNA biallelic transcription

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0033] This Example 1 provides how to apply the method of the present invention to the targeted knockout of the MALAT1 gene. Among them, MALAT1 (located in the first intron of MALAT1) was selected as the target region, and homologous recombination vectors MALAT1-CMV-PuroR-BGH PolyA and MALAT1-CMV-NeoR-BGH PolyA containing dual antibiotic selection genes were constructed, and CRISPR / Cas9-mediated DNA double-strand break, insert it downstream of the MALAT1 promoter through homologous recombination, terminate the expression of the MALAT1 gene, and finally use double antibiotic screening genes for efficient screening to obtain cell lines with simultaneous knockout of both alleles.

[0034] According to different target genes, this method can be applied to different target genes including human by designing and modifying the corresponding homology arm sequence of the desired target gene and the gRNA sequence (of the gene targeted by CRISPR / Cas). Species cell line gene transcriptio...

Embodiment 2

[0075] This example 2 provides the optimized comparison of transcription termination signal SV40PolyA (Simian vacuolating virus40PolyA), BGH PolyA (bovine growth hormone PolyA) and β-globin teminator in the present invention in terms of termination efficiency, molecular weight, and reverse sequence termination efficiency (See figure 1 ). Among them, SV40 PolyA (Simian vacuolating virus 40PolyA) and BGH PolyA (bovine growth hormone PolyA) are transcription termination signals that can be widely used in molecules; and β-globin teminator is the termination signal of gene β-globin, which provides a more thorough research on transcription Termination signal. Using these three transcription termination signals, compare their termination efficiencies, molecular weights, and reverse sequence termination efficiencies, see figure 1 .

[0076] Specifically include the following steps:

[0077] a) Design PCR amplification primers for SV40PolyA, β-globin teminator and BGH PolyA. SV40P...

Embodiment 3

[0091] In Example 3, monoclonal cell lines were obtained from the polyclonal cell lines obtained in Example 1 by using the limiting dilution method, and the transcription of MALAT1 was detected by RT-qPCR.

[0092] Specifically include the following steps:

[0093] a) When using the limiting dilution method to obtain a monoclonal cell line, firstly dilute the cells obtained after screening in Example 1. About 50 cells in 10ml medium, inoculate 100μl in each well of a 96-well plate, and culture for 15 days;

[0094] b) DNA and RNA were extracted according to the instructions of TRIzol reagent (purchased from Life Company);

[0095] c) Using the genotyping PCR method, the upstream and downstream primers of the homologous recombination region such as Figure 7 As indicated, the extracted sample DNA was used as a template for PCR detection ( Figure 8 ). The nucleotide sequences of the primers used in this detection method are as follows:

[0096] MALAT1-HDR-F: 5'-CCCTCGGAGTT...

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Abstract

The invention provides a method for terminating lncRNA diallele transcription. The method comprises the following steps of 1) constructing CMV-antibiotic screening gene-PolyA DNA fragments containing two different antibiotic screening genes; 2) designing gRNA according to different target genes, and constructing Cas9 / gRNA expression vectors; 3) inserting the CMV-antibiotic screening gene-PolyA DNA fragments obtained in the step 1) into the target genes, and constructing homologous recombinant vectors containing the target genes; 4) introducing cells: co-transfecting the pair of homologous recombinant vectors containing the target genes obtained in the step 3) and the Cas9 / gRNA expression vectors containing the target genes obtained in the step 2) to the cells; and 5) performing screening for at least 2 weeks by using related antibiotics used in the step 1), and obtaining polyclonal cell strains of which lncRNA dialleles are knocked out. According to the method, the purpose of quickly obtaining the cell strains of which the lncRNA dialleles are knocked out at the same time while realizing efficient fixed-point insertion can be achieved.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for terminating long-chain non-coding RNA biallelic gene transcription. Background technique [0002] The study of the function of long non-coding RNA (lncRNA) is becoming a hot spot, and the loss of gene expression plays a very important role in the study of gene function. The conventional strategy to cause loss of gene expression is RNA interference. However, due to the high off-target rate and incomplete down-regulation of RNA, the wide application of this method is largely limited. Although frameshift mutations of coding genes mediated by gene editing tools, or large fragment deletions of entire genes or promoters can effectively achieve the purpose of gene knockout or knockdown. However, frameshift mutations cannot effectively knock out lncRNAs. At the same time, in the study of long non-coding RNA genes, the deletion of large fragments may lead to the loss of ot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85
CPCC12N15/113C12N15/85C12N2310/10C12N2800/107C12N2800/60C12N2800/80C12N2810/10C12N2830/50
Inventor 崔恒宓刘洋洋
Owner YANGZHOU UNIV
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