MicroRNA structure-based construction method and function verification of hepatic cell selective multi-target interfering plasmid vector

A technology of plasmid vectors and hepatocytes, applied in the field of molecular biology, can solve the problems of reduced interference efficiency, no cell/tissue specificity, easy methylation, etc.

Active Publication Date: 2012-05-02
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Transient overexpression of shRNA can easily lead to the blockage of intracellular microRNA pathway and cause cytotoxicity
There are also people who use the structure of microRNA to construct multi-target shRNA expression vectors (Hyun Jung Junn, Dai-Wu Seol et al. Effective knockdown of multiple target genes by expression the single transcript harboring multi-cistronic shRNAs. Biochemical and Biophysical Research Communications 396 (2010 )861-865), but this vector uses a CMV promoter, which can initiate the transcription of shRNA in a variety of cells, inhibit the expression of target genes, has no cell / tissue specificity, and is prone to methylation and inactivation, resulting in reduced interference efficiency

Method used

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  • MicroRNA structure-based construction method and function verification of hepatic cell selective multi-target interfering plasmid vector
  • MicroRNA structure-based construction method and function verification of hepatic cell selective multi-target interfering plasmid vector
  • MicroRNA structure-based construction method and function verification of hepatic cell selective multi-target interfering plasmid vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Hepatocyte selective multi-target interference plasmid vector pLIVE-(shLuc155 based on microRNA structure mir -shLuc155 mir -shEGFP131 mir -shEGFP425 mir ) construction

[0053] 1. Transformation of pLIVE plasmid (Mirus company)

[0054] (1) Remove Xba I, Spe I restriction endonuclease sites:

[0055] The Xba I and Spe I sites in the pLIVE initial plasmid were removed by filling in the 5' protruding ends and self-ligating. First, 2 μg of the pLIVE initial plasmid was cut with Spe I enzyme (purchased from Fermentas, Cat.#FD1254), and the digested product was analyzed by agarose gel electrophoresis, and the linear pLIVE plasmid was recovered, and Klenow Fragment (purchased from Fermentas, Cat.# EP0052) to fill in the 5' protruding end, and then use T4 ligase (purchased from Fermentas, Cat. #EL0011) to ligate the linear plasmid to form a circular shape, and the newly generated sequence after ligation does not contain a Spe I site. The ligation product was ...

Embodiment 2

[0100] Example 2: Functional identification of hepatocyte-selective single-target, dual-target, and multi-target interference plasmid vectors targeting reporter genes Luciferase and EGFP

[0101] 1. The constructed liver cell selective single-target interference plasmid pLIVE-shLuc155 mir , dual-target interference plasmid pLIVE-(shLuc155 mir -shLuc155 mir ) was transformed into Escherichia coli DH5α and amplified, the cells were collected by centrifugation, and the medium was prepared to extract and purify the plasmid, and the OD was determined 260 Plasmid concentration, sequencing confirmed that the plasmid sequence was correct.

[0102] 2. Introducing hepatocyte-selective single-target interference plasmid pLIVE-shLuc155 mir , dual-target interference plasmid pLIVE-(shLuc155 mir -shLuc155 mir ) and the Luciferase expression plasmid pGL3-CMV (purchased from Promega, Cat. #E1751, inserted into the CMV promoter) at a ratio of 3:1, co-transfected with liposome lipofectamin...

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Abstract

The invention discloses a microRNA structure-based construction method and function verification of a plasmid vector pLIVE-(shLuc155mir-shLuc155mir-shEGFP131mir-shEGFP425mir) capable of realizing hepatic cell selective multi-target interfering. The plasmid vector consists of an alpha-fetoprotein (AFP) enhancer / albumin promoter, a multiple cloning site (MCS) region, two introns, and microRNA30-based shRNA (ShRNAmir) transcription units of a plurality of target genes and a plasmid sequence. In the region of the second intron 2, through the design of an isocaudarner structure, a plurality of identical or different shRNAmir transcription units for a luciferase gene and a green fluorescent protein (EGFP) gene are connected in series, and at the same time, the expression of the luciferase gene and the expression of the EGFP gene in hepatic cells are inhibited selectively with high efficiency. The vector and the construction method thereof can be used in researches on functions of liver cell genes and development of gene treatment medicines of liver diseases, and have high reverence value of construction of specific interfering vectors of other cells / tissues.

Description

technical field [0001] The invention relates to a plasmid vector pLIVE-(shLuc155 mir -shLuc155 mir -shEGFP131 mir -shEGFP425 mir ) construction method and application thereof, belonging to the field of molecular biology. technical background [0002] RNA interference (RNAi) is a post-transcriptional gene silencing phenomenon induced by double-stranded RNA that is highly conserved during evolution. The phenomenon of RNAi was first discovered in the study of Caenorhabditis elegans by Fire et al. in 1998, and since then it has been confirmed that RNAi also exists in fungi, Drosophila, Arabidopsis and mammalian cells. At present, RNAi technology has made remarkable progress in a wide range of fields such as functional genomics research, microbiology research, gene therapy and signal transduction, making its application in the medical field have broad prospects. [0003] When exogenous genes such as viral genes, artificially transferred genes or transposons enter the host ce...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/63
Inventor 田志刚耿建林孙汭魏海明
Owner UNIV OF SCI & TECH OF CHINA
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