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Construction of siRNA and its expression vector targeting mouse ucp2 gene expression

A technology of gene expression and RNA interference, applied in DNA/RNA fragments, gene therapy, genetic engineering, etc., can solve the problem of low infection efficiency and achieve the effect of improving transfection efficiency and facilitating transfection efficiency

Inactive Publication Date: 2018-07-17
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first two methods have low infection efficiency, especially for cells with poor interference effect of liposomes, such as neural stem cells

Method used

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  • Construction of siRNA and its expression vector targeting mouse ucp2 gene expression
  • Construction of siRNA and its expression vector targeting mouse ucp2 gene expression
  • Construction of siRNA and its expression vector targeting mouse ucp2 gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Design of an siRNA sequence that targets and inhibits the expression of the mouse UCP2 gene.

[0031] According to the mRNA sequence of UCP2 gene in mouse neural stem cells (NM_011671), a set of siRNA sequences and control siRNA sequences targeting mUCP2 gene expression were designed, and a siRNA sequence with the best interference effect was screened out, named LVT818.

[0032] The specific siRNA sequence is as follows, wherein LVT818 is an effective interference fragment group, and LVT4 is a negative control group.

[0033]LVT818: 5'-CCTAATGGCTGCCTACCAA-3'; GC52.6%.

[0034] LVT4: 5'-TTCTCCGAACGTGTCACGT-3'; GC52.6%.

Embodiment 2

[0035] Example 2: Design and synthesis of oligonucleotides.

[0036] The sense strand and antisense strand of shRNA were designed and synthesized with the siRNA sequence designed in the above-mentioned Example 1. The Loop structure in the shRNA selected "cTCAAGAGA" and "TCTCTTGAg", and added at the 5' end respectively Age I and EcoR I The sticky end of the restriction site is synthesized by Invitrogen, and the specific shRNA sequence is as follows.

[0037] LVT818-1 (justice chain):

[0038] 5'- Ccgg CCTAATGGCTGCCTACCAAcTCAAGAGATTGGTAGGCAGCCATTAGG TTTTTTg -3'.

[0039] LVT818-2 (antisense strand):

[0040] 5'- aattcaaaaaa CCTAATGGCTGCCTACCAATCTCTTGAgTTGGTAGGCAGCCATTAGG-3'.

[0041] LVT4-1 (justice strand):

[0042] 5'-CcggTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTg-3'.

[0043] LVT4-2 (antisense strand):

[0044] 5'-aattcaaaaaTTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAA-3'.

[0045] After the shRNA sequence synthesized above was dissolved to 20 μM ...

Embodiment 3

[0046] Example 3: Construction of an interfering lentiviral vector for targeted inhibition of mouse UCP2 gene expression.

[0047] Schematic diagram of the structure of the lentiviral vector pMagic 4.1 figure 1 . The vector contains a U6 promoter, which can continuously promote the expression of downstream genes in host cells and continuously express small RNAs with interfering effects. The vector also contains a CMV promoter, which can drive the expression of the fluorescent protein EGFP, which facilitates the detection of transfection efficiency and infection efficiency.

[0048] by Age I and EcoR I Double-enzyme cut the lentiviral vector pMagic 4.1 to make it linear, after gel purification and recovery, use T4 ligase to connect with the annealed product of Example 2 overnight at 16°C, transform competent bacteria, pick recombinant positive clones, after transformation and screening, Sent to Invitrogen Company for sequencing identification.

[0049] (Positive clone se...

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Abstract

The invention relates to an RNA interfering recombinant lentiviral vector for targeted inhibition of mouse neural stem cell UCP2 gene expression and construction thereof. The construction comprises the steps of firstly constructing a recombinant lentiviral vector pNL-EGFP / CMV / WPREdU3-sh mUCP2 aiming at a mouse neural stem cell UCP2 gene, and then co-transfecting a 293T cell by using the recombinant lentiviral vector together with a second-generation lentiviral packaging plasmid pCD / NL-BH*DDD and a membrane protein expression plasmid pLTR-G to obtain the packaged recombinant lentiviral vector. By transfecting a mouse primary neural stem cell by using the recombinant lentiviral vector obtained by the invention, the mouse neural stem cell UCP2 gene expression can be efficiently and specifically inhibited, and a good experimental foundation is laid for further functional researches on the mouse UCP2 gene.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to an RNAi recombinant lentiviral expression vector for mouse neural stem cell UCP2 gene and the construction of the expression vector. The recombinant lentivirus described in the present invention can be applied to the research on the pathogenesis of neural tube defects. Background technique [0002] Neural Tube Defects (NTDs) refer to congenital birth defects with the central nervous system as the main site of disease. Shanxi Province, my country has the highest incidence rate, with a statistical result of 19.9‰. The high incidence of NTDs has become the main cause of lifelong disability and death of infants in many countries and regions around the world. [0003] The closing process of the neural tube is tightly controlled by genes as well as regulated by environmental factors. According to the applicant's research results and literature search in recent years, the relatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/867C12N15/66A61K48/00A61P25/00
Inventor 刘志贞解军张引红胡俊加三三于娟刘丹郭倩魏秀丽陈欢直索金荣
Owner SHANXI MEDICAL UNIV