Method for constructing tandem expression small interfering RNA recombinant lentiviral vector

A lentiviral vector and recombinant lentivirus technology, applied in the field of molecular biology, can solve the problems of silencing multiple target genes at the same time, which is not suitable, and achieve the effect of preventing or delaying virus escape, avoiding loss, and easy directional insertion.

Inactive Publication Date: 2010-03-31
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above methods have their own advantages and disadvantages: the siRNAs or siRNA expression cassettes prepared by the first four methods can be transfected or electroporated into cells, which can achieve transient gene silencing, but are not suitable for proteins with a long half-life.
[0005] However, the siRNA lentiviral expression vector based on the type III promoter that has been developed is only suitable

Method used

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  • Method for constructing tandem expression small interfering RNA recombinant lentiviral vector
  • Method for constructing tandem expression small interfering RNA recombinant lentiviral vector
  • Method for constructing tandem expression small interfering RNA recombinant lentiviral vector

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Promoter modification of lentiviral vector

[0045] The lentiviral vector modified by the applicant is pLentiLox 3.7 (Rubinson and Dillon, Nature Genetics, 2003). The vector contains a mouse U6 promoter to control the expression of siRNA, and also contains a reporter gene EGFP for flow cytometry sorting. In order to avoid homologous recombination caused by a single promoter of the tandem siRNA, resulting in the loss of the siRNA expression box, the applicant replaced the mouse U6 promoter of the vector with a human U6 promoter and a human H1 promoter (human U6 promoter and the human H1 promoter were obtained from Ambion's products pSilencer2.0_U6 and pSilencer3.0_H1 by PCR), and the transformed lentiviral vectors were named pLLU6 and pLLH1 respectively (Escherichia coli Stbl3 / pLLU6 CCTCC M209135; Escherichia coli Stbl3 / pLLH1 CCTCC M 209136). For the schematic diagram of promoter transformation, see figure 1 shown. The specific transformation process is a...

Embodiment 2

[0061] Example 2: Construction method of tandem expression siRNA recombinant lentiviral vector

[0062] 2.1 Construction of single-target siRNA recombinant lentiviral vector (see figure 2 Step I)

[0063] 2.1.1 Chemically synthesized oligonucleotide chains, the format is as follows:

[0064] Sense strand: 5'-(19N)-(TTCAAGAGA)-(N91)-TTTTTTTC-3'

[0065] Antisense strand: Complementary to the sense strand with TCGA added at the 5' end to create Xho1 cohesive overhangs

[0066] Note: 1) 19N represents the 19-base sequence of the siRNA target, and N19 represents the reverse complementary sequence of 19N;

[0067] 2) (TCAAGAGA) guides the generation of the "loop" region of the siRNA precursor shRNA (small hairpin RNA), which sequence is selected from (Brummelkamp et al., Science, 2002)

[0068] 2.1.2 Anneal the sense strand and antisense strand to form a fragment with Xho1 sticky end and blunt end;

[0069] 2.1.3 Ligate the above fragments to the vector pLLU6 or pLLH1 (the ve...

Embodiment 3

[0092] Example 3: Application example of lentiviral vector expressing siRNA in tandem

[0093] In order to verify whether the lentiviral vector expressing siRNA in tandem can work normally, the applicant explained with an application example of interfering with HIV:

[0094] In this example, the applicant selected the HIV genes rev, pol, tat and the host cell membrane protein CCR5 required for virus-infected cells as targets, and used the lentiviral vector for tandem expression of siRNA provided by the present invention to construct a single target Point, double-target, triple-target siRNA recombinant lentiviral vector. The combinations of siRNA expression cassettes are shown in the table below:

[0095] single target

H1 CCR5

U6 rev

U6 pol

U6

double target

H1 CCR5_U6 rev

H1 CCR5_U6 pol

H1 CCR5_U6 tat

Three targets

H1 CCR5_U6 rev_U6 pol

H1 CCR5_U6 rev_U6 tat

H1 CCR5_U6 pol_U6 tat ...

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Abstract

The invention discloses a method for constructing a tandem expression small interfering RNA recombinant lentiviral vector. The method comprises the following steps of: (1) constructing the siRNA recombinant lentiviral vector with single target spot: a, chemically synthesizing an oligonucleotide chain; b, annealing a sense chain and an anti-sense chain; and c, connecting an annealing fragment to the vector; (2) constructing the siRNA recombinant lentiviral vector with double target spots: a, amplifying a U6/H1-siRNA expression frame from the siRNA recombinant lentiviral vector with single target spot by utilizing the PCR; b, connecting the expression frame to the siRNA recombinant lentiviral vector with single target spot undergoing enzyme digestion by the XhoI; and c, performing the enzymedigestion, identification and screening of the clone forwardly inserted in the expression frame; and (3) constructing the siRNA recombinant lentiviral vector with multiple target points in tandem connection: inserting the U6/H1-siRNA expression frame in the siRNA recombinant lentiviral vector with double target spots to construct the siRNA recombinant lentiviral vector with triple target spots, and obtaining the siRNA recombinant lentiviral vector with multiple target points in tandem connection by repeating the step. The construction method is suitably used for the expression of the siRNA with multiple target genes as well as the research on the multiple target genes which are silent for a long time.

Description

technical field [0001] The present invention relates to the field of molecular biology. More specifically, it relates to a method for constructing a recombinant lentiviral vector expressing small interfering RNA in tandem. Background technique [0002] RNA interference (RNA interference, RNAi) refers to the process of sequence-specific post-transcriptional gene silencing (post-transcriptional gene silencing, PTGS) triggered by double-stranded RNA. At present, the mechanism of RNA interference has been basically clarified: endogenous or exogenous double-stranded RNA is cut by Dicer in the cytoplasm into small interfering RNA (siRNA) of 21-23 nt with 2 bases protruding from the 3' end, which is related to RNA-induced silencing complex (RNA-induced silencing complex, RISC) binding. The siRNA is melted in RISC, where the antisense strand guides RISC to target its complementary mRNA, and RISC cleaves the mRNA in the middle of the complementary sequence, thereby inhibiting the e...

Claims

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Application Information

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IPC IPC(8): C12N15/867
Inventor 郭德银柳叶陈宇申彦森
Owner WUHAN UNIV
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