Activated siRNA able to enhance gene expression

A gene expression and gene technology, applied in the field of activating siRNA, can solve the problems of inability to be widely used, elevated genes, and few reports, and achieve the effects of great application value, enhanced expression, and broad application prospects.

Active Publication Date: 2014-07-23
GUANGZHOU QIANYANG BIO-TECH PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the method of trying to up-regulate genes is mainly through the artificial design of transcription factor mimics, but like endogenous transcription factors, it will cause the expression of a large number of downstream target genes and cannot specifically increase the expression of a certain gene

Method used

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  • Activated siRNA able to enhance gene expression
  • Activated siRNA able to enhance gene expression
  • Activated siRNA able to enhance gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Preparation of plasmids, microRNA mimics, siRNA, single-stranded RNA (ssRNA)

[0051] The CMV promoter on the pMIR-Reporter vector (Promega) was replaced with the -400~+1bp segment near the transcription start site (TSS) of the human IL-2 promoter with restriction enzymes to construct the human IL-2 promoter promoter luciferase reporter vector. The same method was used to construct reporter vectors containing promoters of HIV-1, human INS, human APOE, and other genes. Hsa-let-7i mimics, ssRNA and corresponding negative controls were purchased from Shanghai Genepharma. The siRNA targeting the TATA-box motif of the gene promoter was purchased from Ribobio, Guangzhou.

[0052] cell culture

[0053] Jurkat and HEK293T cell lines were purchased from ATCC and cultured according to its operating specifications. Human peripheral blood lymphocytes (PBMCs) were isolated from healthy human whole blood with lymphocyte separation medium, and then human CD4 + T Cell Isolation K...

Embodiment 2

[0054] Example 2 Optimization of siRNA

[0055] According to the experimental results of continuous advancement, siRNA targeting the TATA-box motif of the gene promoter was purchased from Guangzhou Ribobio in batches:

[0056] Group 1: Design different siRNAs according to the position of the TATA-box motif;

[0057] Group 2: optimize the sequence at the 5' end of the siRNA antisense strand targeting the TATA-box motif of the IL-2 promoter as the center: make the first base U, or make the first two bases Base is UA;

[0058] Group 3: On the basis of the siRNA sequences of Group 2, the following optimizations were performed: the sequence lengths were 15, 17, 19, 21, 23, 25, 27 or 29 nucleotides;

[0059] Group 4: According to Figure 4 Instructions, chemical modification of siRNA targeting the TATA-box motif of the IL-2 promoter.

[0060] Group 5: According to Figure 5 Instructions, mutation design was performed on siRNAs targeting the TATA-box motifs of IL-2 and INS promo...

Embodiment 3

[0062] Example 3 Transfection

[0063] According to the product instructions, the above groups of optimized siRNA and the corresponding luciferase reporter plasmid were co-transfected into HEK293T cells with Lipofectamine 2000 (Invitrogen).

[0064] Optimized siRNA transfection into Jurkat cell line, human or mouse CD4 using Lipofectamine RNAiMAX (Invitrogen) + T cells were adjusted to a final concentration of 100-200 nM. After 12-24 hours, the Jurkat cell line was stimulated with PMA (50 ng / ml, Sigma) and ionomycin (1 μM, Sigma) for 48-72 hours, and anti- CD3 (1μg / ml, R&D Systems) and anti-CD28 (5μg / ml, R&D Systems) combined human CD4 + T cells were stimulated for 48-72 hours, and mouse CD4 + T cells were stimulated for 48-72 hours.

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Abstract

The invention discloses an activated siRNA able to specifically enhance gene expression through a gene promoter with a TATA-box motif in the target. The activated siRNA has the characteristics that: (1) the activated siRNA is complementary with a position taking the TATA-box motif as the center; (2) the first two base groups of an antisense strand adopt UA; (3) the length is 23 nucleotides; (4) methylation modification is conducted on the 3' end of the antisense strand; and (5) mismatch of the 3' end of the antisense strand is avoided. The activated siRNA provided by the invention can significantly enhance the target expression for a long time, and has great application value and broad application prospects in biotechnology, gene therapy drug development and other fields.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and biomedicine, and more specifically relates to an activating siRNA that specifically enhances gene expression by targeting a gene promoter with a TATA-box motif. Background technique [0002] In the genomes of plants and animals, a large number of genes express non-coding RNA (non-coding RNA, ncRNA), such as small interfering RNA (small interfering RNA, siRNA), microRNA (microRNA, miRNA), ribosomal RNA (ribosomal RNA, rRNA) and transfer RNA (transfer RNA, tRNA), etc. Non-coding RNA plays an important regulatory role in the transcription and post-transcriptional levels of genes, and even in the process of animal development and disease occurrence. The RNA interference (RNA interference, RNAi) technology, which uses double-stranded RNA molecules homologous to the target gene to inhibit the expression of the target gene, has a wide range of applications in the fields of functional geno...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K48/00
Inventor 张辉张意军樊苗苗
Owner GUANGZHOU QIANYANG BIO-TECH PHARM CO LTD
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