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Construction method for vector for efficiently expressing small RNA and expressing multiple small RNAs by using cynomolgus monkey U6 promoter

A construction method and promoter technology, applied in the field of genetic engineering, can solve problems such as the reduction of cloning efficiency, and achieve the effect of simple method and high-efficiency transcription ability

Active Publication Date: 2020-05-01
YUNZHOU BIOSCIENCES (GUANGZHOU) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using this method, up to 9 fragments can be cloned into the target vector at the same time, and the positive rate of PCR identification reaches more than 70% in the cloning of 2 to 3 fragments. As the number of cloned fragments increases, the cloning efficiency gradually decreases

Method used

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  • Construction method for vector for efficiently expressing small RNA and expressing multiple small RNAs by using cynomolgus monkey U6 promoter
  • Construction method for vector for efficiently expressing small RNA and expressing multiple small RNAs by using cynomolgus monkey U6 promoter
  • Construction method for vector for efficiently expressing small RNA and expressing multiple small RNAs by using cynomolgus monkey U6 promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Obtaining macU6 promoter information:

[0042] Using the known human and mouse U6 promoters as templates, the cynomolgus monkey (Macafascicularis) genome was screened on NCBI for Blast homology comparison (such as figure 1 As shown, wherein TTATATA is the TATA box of the macU6 promoter), the reference sequence of the macU6 promoter was obtained, and its DNA sequence is shown in SEQ ID NO:1.

Embodiment 2

[0043] Example 2 Construct a vector for the expression of a single gRNA mediated by a macU6 promoter, and verify its targeting efficiency at the cellular level:

[0044] 1 Vector construction:

[0045] 1.1 Construction of pLV[gRNA]-hPGK>EGFP:T2A:Puro-macU6>ROSA26[gRNA#1]

[0046] SpeI digested pLV[gRNA]-EGFP:T2A:Puro-U6>AAVS1-gRNA3 (commodity ID is VB191223-3660qkw, please log in https: / / www.vectorbuilder.cn / for details), and recovered a 7.7kb vector backbone fragment ; Using the cynomolgus monkey genome as a template, use primers SpeI-macU6-F and SpeI-macU6-ROSA26[gRNA#1]-R PCR amplification, reclaim about 300bp SpeI-macU6 target fragment (such as figure 2 shown); using pLV[gRNA]-EGFP:T2A:Puro-U6>AAVS1-gRNA3 as a template, using primers ROSA26[gRNA#1]-Scaffold-hPGK-F and SpeI-hPGK-R PCR amplification, recovery of about The 430bp ROSA26[gRNA#1]-Scaffold-hPGK target fragment; in the primer sequence, italic indicates the homology arm sequence used in the subsequent gibson re...

Embodiment 4

[0108] Example 4 Construct the vector of shRNA interference mediated by the macU6 promoter, and verify its interference efficiency at the cellular level:

[0109] 1. The present invention adopts macU6 promoter to express shRNA, and the interfering gene is EGFP. Construct the following two interference vectors:

[0110] Vector 1: pLV[shRNA]-hPGK>Puro-macU6>EGFP[shRNA]

[0111] Vector 2: pLV[shRNA]-hPGK>Puro-macU6>Scramble_shRNA

[0112]1.1 Enzyme digestion and ligation: First, use AgeI and EcoRI to digest the vector backbone pLV.shRNA(macU6).P / Puro (the product ID is VB191226-1031aup, please log in to https: / / www.vectorbuilder.cn / for details), and recover the vector The large fragment of the backbone is then ligated with EGFP[shRNA] and Scramble_shRNA annealing products respectively to obtain the above-mentioned carrier 1 and carrier 2, wherein carrier 1 is an interference carrier for EGFP, and carrier 2 is a negative control carrier without interference effect. The sequenc...

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Abstract

The invention provides a cynomolgus monkey U6 gene promoter macU6. The cynomolgus monkey U6 gene promoter macU6 comprises a nucleotide acid sequence shown as SEQ ID NO: 1. A segment of sequence homologous with the U6 promoter, namely the macU6 promoter, is obtained in a cynomolgus monkey genome for the first time, and the efficient transcription capability is achieved. And the macU6 promoter is introduced into a multi-gRNA expression vector. According to the method, by adoption of a method of combining a Golden Gate cloning technology and a Gateway recombination cloning technology, the multi-gRNA expression vector with gRNA tandem expression driven by different U6 promoters is constructed; and the method is simple, efficient, flexible and time-saving. Meanwhile, by introduction of a novelhighly-efficient macU6 promoter, the problem of vector instability caused by too few types of U6 promoters and repeated occurrence of U6 is solved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a cynomolgus monkey U6 gene promoter and its cloning and application. Background technique [0002] In eukaryotes, three types of RNA polymerase (PolI, PolII and PolIII) promoters are mainly responsible for the transcription of different types of genes, among which PolIII promoters are widely used in the expression of small RNAs because of their ability to efficiently transcribe small RNAs, including Small interfering RNA, short hairpin RNA (shRNA) and gRNA in CRISPR-Cas systems for RNAi applications. The most widely used and most efficient Pol III promoters include human U6 promoter (hU6), mouse U6 promoter (mU6) and human H1 promoter (hH1). Studies have shown that the transcription of the U6 promoter starts precisely from the first A or G within the range of the transcription site -1 to +2. Based on this feature, people have modified the U6 promoter by ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867C12N15/66
CPCC07K14/47C12N15/66C12N15/86C12N2740/15043
Inventor 蓝田施金秀罗燕
Owner YUNZHOU BIOSCIENCES (GUANGZHOU) INC
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