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A kind of lentiviral vector and application thereof for expressing lncrna

A lentiviral vector and plasmid technology, applied in the field of genetic engineering, can solve the problems of low transfection efficiency and difficulty in building stable cell lines, achieve high sensitivity, reduce the number of experimental animals, and avoid damage

Active Publication Date: 2016-03-30
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problems of low transfection efficiency and difficulty in establishing stable cell lines in the above-mentioned lncRNA expression vectors, a lentiviral vector added with human EF-1α promoter sequence and SV40polyA transcription termination signal sequence was constructed to achieve stable ectopic expression of lncRNA. The invention is realized like this:

Method used

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  • A kind of lentiviral vector and application thereof for expressing lncrna
  • A kind of lentiviral vector and application thereof for expressing lncrna
  • A kind of lentiviral vector and application thereof for expressing lncrna

Examples

Experimental program
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Effect test

Embodiment 1

[0046] The construction of embodiment 1 lentiviral vector

[0047] 1. Construction of pLL3.7-luc2

[0048] 1.1 PCR reaction:

[0049] Using the pmirGLO plasmid as a template, the upstream primer SEQIDNO.1 and the downstream primer SEQIDNO.2 amplify luc2;

[0050]PCR reaction system 40 μL, including TaqPlusDNA Polymerase 2 μL, Mastermix 12 μL, pmirGLO plasmid 2 μL, 10 μmol / LSEQ ID NO.12 μL, 10 μmol / LSEQ ID NO.22 μL, ddH 2 O20 μL;

[0051] PCR reaction program: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 60s, a total of 30 cycles; renaturation at 72°C for 10min;

[0052] 1.2 Gel electrophoresis:

[0053] Perform agarose gel electrophoresis on the PCR product, 120V, 30min, according to the instructions, use the rapid DNA product purification kit to recover the target fragment, and obtain the purified PCR product;

[0054] 1.3 Construction of cloning vector

[0055] Add 5 μL of the purified PCR product obt...

Embodiment 2

[0077] Example 2 pLnc-luc2 plasmid expresses LncRNAMEG3

[0078] LncRNAMEG3 plasmid (NR_002766.2 in NCBI) was synthesized by GenScript Biotechnology Co., Ltd.

[0079] 1. Carry out BamHI and XhoI double enzyme digestion to the LncRNAMEG3 plasmid and the pLnc-luc2 plasmid obtained in Example 1, and the reaction system is as follows:

[0080] LncRNAMEG3 plasmid 6μL (260ng / μL)+10xbuffer2μL+BamHI1μL+XhoI1μL+ddH 2 O 10 μL;

[0081] pLnc-luc2 plasmid 10μL (320ng / μL)+10xbuffer2μL+BamHI1μL+XhoI1μL+ddH 2 06 μL;

[0082] The above reaction systems were placed at 37°C for 4 hours, and then agarose gel electrophoresis was carried out at 120V for 30 minutes. After electrophoresis, the AxyPrepDNA gel recovery kit was used to recover the electrophoresis product according to the instructions, and the purified product and Purified product after pLnc-luc2 plasmid digestion;

[0083] Identification: Add 5 μL ncRNAMEG3 DNA digested purified product, 1 μL 10X Ligationbuffer, 3 μL pLnc-luc2 pl...

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Abstract

The invention discloses a lentiviral vector for expressing lncRNA (long noncoding ribonucleic acid) and application thereof. The nucleotide sequence of the lentiviral vector is shown as SEQIDNO.5, pLL3.7 plasmid is taken as the skeleton, luc2 gene substitutes for the EGFP (enhanced green fluorescent protein) gene of the plasmid, and a synthetic sequence with the nucleotide sequence of SEQIDNO.4 substitutes for an mU6 promoter sequence. The lentiviral vector for lncRNA ectopic expression does not generate any effective cellular immunologic response, can infect the cell in non division stage and contain large exogenous gene segment, transgenosis expression can last for months, and luc2 gene is introduced without using exogenous exciting light, thus preventing normal cells in the body from being damaged, and the lentiviral vector is beneficial for observing for long time.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a lentiviral vector for expressing lncRNA and its application. technical background [0002] Long noncoding RNA (long noncoding RNA, hereinafter referred to as lncRNA) is a type of noncoding RNA (ncRNA) that is longer than 200 nt and does not exhibit any protein-coding potential. LncRNA is structurally similar to messenger RNA (mRNA), but there is no open reading frame (open reading frame, ORF) in the sequence. Many known lncRNAs are transcribed by RNA polymerase (RNApolymerase II, RNApol II) and formed by alternative splicing, usually polyadenylated. The transcription level of lncRNA genes is lower than that of protein-coding genes, widely exists in intergenic introns and antisense strands of protein-coding genes, some overlap with protein-coding genes or non-coding genes, and some are preferentially transcribed only in specific tissues . Kapranov et al. proposed that in hu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867
Inventor 陈云林喆孙倍成唐俊伟万昕卓晗夏阳
Owner NANJING MEDICAL UNIV
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