Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Lentiviral vector for expressing lncRNA (long noncoding ribonucleic acid) and application thereof

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 facilitate long-term observation

Active Publication Date: 2014-06-04
NANJING MEDICAL UNIV
View PDF4 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problems of low transfection efficiency and difficult construction of stable cell lines in the above-mentioned lncRNA expression vectors, a lentiviral vector added with human EF-1α promoter sequence and SV40 polyA transcription termination signal sequence was constructed to achieve stable ectopic expression of lncRNA. The present invention is achieved like this:

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Lentiviral vector for expressing lncRNA (long noncoding ribonucleic acid) and application thereof
  • Lentiviral vector for expressing lncRNA (long noncoding ribonucleic acid) and application thereof
  • Lentiviral vector for expressing lncRNA (long noncoding ribonucleic acid) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of lentiviral vector

[0048] 1. Construction of pLL3.7-luc2

[0049] 1.1 PCR reaction:

[0050]Using the pmirGLO plasmid as a template, the upstream primer SEQ ID NO.1 and the downstream primer SEQ ID NO.2 amplify luc2;

[0051] PCR reaction system 40 μL, including Taq Plus DNA Polymerase 2 μL, Master mix 12 μL, pmirGLO plasmid 2 μL, 10 μmol / L SEQ ID NO.1 2 μL, 10 μmol / L SEQ ID NO.2 2 μL, ddH 2 O 20 μL;

[0052] PCR reaction program: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 60 s, a total of 30 cycles; renaturation at 72°C for 10 min;

[0053] 1.2 Gel electrophoresis:

[0054] 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;

[0055] 1.3 Construction of cloning vector

[0056] Add 5 μL of the purified PCR...

Embodiment 2

[0079] Example 2 pLnc-luc2 plasmid expresses LncRNA MEG3

[0080] LncRNA MEG3 plasmid (NR_002766.2 in NCBI) was synthesized by GenScript Biotechnology Co., Ltd.

[0081] 1. Carry out BamH I and Xho I double enzyme digestion to LncRNA MEG3 plasmid and the pLnc-luc2 plasmid that embodiment 1 obtains, and reaction system is as follows:

[0082] LncRNA MEG3 plasmid 6μL (260ng / μL)+10x buffer 2μL+BamH I 1μL+Xho I 1μL+ddH 2 O 10 μL;

[0083] pLnc-luc2 plasmid 10 μL (320ng / μL)+10x buffer 2μL+ BamH I 1μL+Xho I 1μL+ddH 2 O 6 μL;

[0084] Place the above reaction system at 37°C for 4 hours, then perform agarose gel electrophoresis at 120V for 30 minutes. After electrophoresis, use the AxyPrep DNA Gel Recovery Kit to recover the electrophoresis product according to the instructions to obtain LncRNA MEG3 after DNA digestion Purified product and pLnc-luc2 plasmid digested purified product;

[0085] Identification: Add 5 μL ncRNA MEG3 DNA digested purified product, 1 μL 10X Lig...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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 (RNA polymerase II, RNA polII) 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 h...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com