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Construction and application of RNA nuclear retention vector

An expression vector, cell nucleus technology, applied in the field of life science nucleic acid research, can solve problems such as wrong conclusions, hinder the normal function of lncRNA, and achieve the effect of speeding up research

Active Publication Date: 2018-05-04
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even some endogenous lncRNAs localized only in the nucleus, but when expressed exogenously with these vectors, they are often transported to the cytoplasm, thereby hindering the normal function of lncRNAs and leading to erroneous conclusions

Method used

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  • Construction and application of RNA nuclear retention vector
  • Construction and application of RNA nuclear retention vector
  • Construction and application of RNA nuclear retention vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction and expression verification of pZW1-snoVector vector

[0035] According to the molecular structure and production mechanism of sno-lncRNA, the present invention constructs an RNA expression vector named pZW1-snoVector, and uses Northern blot to verify the expression of similar sno-lncRNA molecules.

[0036] 1. Reagents

[0037] Restriction endonucleases XhoI, EcoRI, BglII, PacI and SacII were purchased from NEB Company of the United States, T4 DNA ligase was purchased from Takara Company of Japan; kanamycin (100mg / ml) was purchased from Shanghai Dingguo Biotechnology Co., Ltd.; Reagent and agarose were purchased from Invitrogen Company in the United States, absolute ethanol was purchased from Shanghai Zhenxing Chemical Plant No. 1, and isopropanol was purchased from Shanghai Chemical Reagent Co., Ltd.; AmpliScribe TM T7 Transcription Kits were purchased from Epicentre, USA, LiCl was purchased from Sigma, USA; X-tremeGENE 9 DNA Transfection Reag...

Embodiment 2

[0101] Example 2: Using mouse lncRNA NEAT1 as an example to study the nuclear retention of pZW1-snoVector expression vector

[0102] NEAT1 is a structural long non-coding RNA that is located in the nucleus and is ubiquitous in a variety of cell lines and tissues in higher mammals. nrb , PSF and PSP1α and other proteins are involved in the formation of nuclear substructure paraspeckles. In this experiment, mouse NEAT1 was used as an example to study the effect of pZW1-snoVector expression vector in retaining exogenously expressed RNA in the nucleus.

[0103] 1. Reagents

[0104] Restriction endonucleases SalI and KpnI were purchased from NEB Company in the United States, and T4 DNA ligase was purchased from Takara Company in Japan. For other reagents, please refer to the reagents in Example 1.

[0105] 2. Cell Lines and Vectors

[0106] Human cervical cancer cell line HeLa, pZW1-snoVector expression vector and pEGFP-C1 (with stopcodon) control vector were used in this experi...

Embodiment 3

[0153] Example 3: Taking different lncRNAs, mRNAs and intron-derived RNAs as examples to further study the nuclear retention of pZW1-snoVector expression plasmids

[0154] In order to further study the nuclear retention effect of pZW1-snoVector expression vector on different lncRNAs and other types of RNA, we added another lncRNA-HOTAIR (GeneID: 100124700, 159bp-2337bp), the coding region (CDS) of Blasticidin resistance gene (SEQ ID NO.30), the coding region (CDS) of SNRPN mRNA (GeneID:8926,466-1188bp), and the second intron (GeneID:283373,462bp) of ANKRD52 and the 4th intron partial sequence (GeneID: 283373, 654bp,) was constructed into pZW1-snoVector expression vector and PEGFP-C1 control vector.

[0155] 1. Reagents

[0156] Restriction endonuclease was purchased from NEB Company of the United States, T4 DNA ligase was purchased from Takara Company of Japan, and other reagents were the same as those in Example 1.

[0157] 2. Cell Lines and Vectors

[0158] Human cervical...

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Abstract

The invention discloses an RNA expression vector capable of retaining exogenously expressed RNA in the nucleus. The RNA expression vector is obtained by transforming the pZW1 plasmid. Specifically, the RNA expression vector is a polyclonal clone of the EGFP sequence in pZW1 It is obtained by inserting SNORD116-13 fragments and SNORD116-14 fragments arranged in sequence from 5' to 3' in the locus region. The invention further discloses the construction method of the RNA expression vector and its application in retaining the exogenously expressed RNA in the nucleus.

Description

technical field [0001] The invention belongs to the field of nucleic acid research in life sciences, and specifically relates to an RNA expression vector capable of retaining exogenously expressed RNA in the nucleus and its preparation and application. Background technique [0002] Long noncoding RNA (long noncoding RNA, lncRNA) is longer than 200 nucleotides, but lacks protein coding ability. lncRNAs can be produced at thousands of sites in the mammalian genome. Many studies have shown that lncRNAs are involved in a series of gene expression regulation and biological processes (see review Ulitsky and Bartel, 2013; Batista and Chang, 2013). So far, it is believed that lncRNAs can recruit, assemble, and modify interacting factors to play a role, such as NEAT1 (Mao et al., 2011), HOTAIR (Rinn et al., 2007; Tsai et al., 2010), ANRIL (Yap et al., 2010), sno-lncRNAs (Yin et al., 2012), etc. Interestingly, many lncRNAs-mediated regulation occurs in the nucleus, so new tools are...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66C12N15/85
Inventor 陈玲玲杨力殷庆飞胡世斌
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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