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Nucleic acid and application thereof

A nucleic acid and target gene technology, which is applied in the direction of nucleic acid carrier, the use of carrier to introduce foreign genetic material, and cells modified by introducing foreign genetic material, etc., to reduce the difficulty of synthesis and increase the effect of down-regulating miRNA

Active Publication Date: 2017-04-26
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, current methods for functional knockout of miRNAs still need to be improved

Method used

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  • Nucleic acid and application thereof
  • Nucleic acid and application thereof
  • Nucleic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Design of miRNA functional down-regulation sequence.

[0036] After clarifying the miRNAs that need to be functionally down-regulated, the binding sequences of the target genes that have been reported or can be predicted are searched, and the binding sequences of the target genes are connected in series.

[0037] Taking miR-1915-3p, which has a high CG content and is difficult to down-regulate functionally, as an example, the individual binding site sequences of target genes are shown in Table 1.

[0038] Table 1:

[0039]

[0040] The sequence designed to functionally down-regulate miR-1915-3p is as follows:

[0041]

[0042] The underline is the miRNA target gene binding site sequence, the underlined bold part is the miRNA target gene complementary pairing sequence, the bold italic part is the recognition site of EcoRI and EcoRV, respectively.

Embodiment 2

[0043] Example 2 Construction of miRNA functional down-regulation plasmid

[0044] 1. According to the design of Example 1, the sequence was synthesized.

[0045] 2. The fragment synthesized in Example 1 was digested with EcoRI and EcoRⅤ by double enzyme digestion ( figure 1 The agarose gel electrophoresis diagram of the fragment synthesized in Example 1 after digestion with EcoRI and EcoR V), and ligate it into the target plasmid (the plasmid map is as figure 1 Shown).

[0046] Ligated into plasmid pCDNA3.1, restriction digestion system is shown in Table 2.

[0047] Table 2:

[0048] Composition Quantity Plasmid 4μg EcoRⅠ10IU EcoRⅤ10IU 10×Buffer 4μl water Add to 40μl

[0049] Mix the ingredients of the digestion system thoroughly and incubate at 37°C for 5 hours

[0050] 3. Purification and recovery

[0051] The digested product was subjected to agarose gel electrophoresis, and purified and recovered with a common DNA product purification kit.

[0052] 4. Connection of plasmids

...

Embodiment 3

[0056] Example 3 Functional verification of miRNA functional down-regulation plasmid

[0057] 1. Establishment of a cell line that stably down-regulates miR-1915-3p

[0058] (1) Human leukemia cells (such as K562 cells or UT-7 cells) are cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), and passaged 1:5 every 3 days. Centrifuge the cells of the leukemia cell line, count them, and resuspend them in medium to a cell density of 3×10 5 Pcs / ml, add 1.5ml per well to the labeled 6-well plate;

[0059] (2) Take 4μg of purified plasmid and fill up to 250μl with Opti-MEM medium (EP tube 1), and mix; take another 1.5ml EP tube, add 240μl Opti-MEM medium and 10μl Lipofectamine 2000 (EP tube 2) ), flick and mix well, let stand at room temperature for 5 min;

[0060] (3) Mix the liquids in EP tubes 1 and 2 and mix gently. After standing at room temperature for 20 minutes, add the corresponding liquid to the cell suspension in the prepared 6-well plate drop by drop, shake...

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Abstract

The invention provides a separated nucleic acid with a miRNA target gene binding site sequence. By the separated nucleic acid in an embodiment, miRNA reduction can be realized. Especially, as for high-CG-content miRNA with CG content being up to 90%, reduction efficiency of the nucleic acid is remarkably improved as compared with that in the prior art.

Description

Technical field [0001] The present invention relates to the field of biology. Specifically, the present invention relates to isolated nucleic acids, methods for down-regulating miRNA in cells, and methods for establishing stable miRNA down-regulating cell lines. Background technique [0002] MicroRNA (miRNA) is a type of single-stranded non-coding RNA produced by the endogenous expression of eukaryotes. miRNA can bind to the 3'UTR region of the mRNA transcribed by the target gene in the form of base complementary pairing. Very few bind to the 5'UTR region or ORF region to degrade the mRNA of the target gene or inhibit its translation, thereby down-regulating the target protein. Expression, to regulate gene expression, the number of target genes that can be regulated by a single microRNA molecule is more than 200, and there is a lot of evidence that miRNA plays a role in hematopoietic differentiation, tumorigenesis, inflammation and other physiological and case conditions. Import...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N5/10
CPCC12N5/0694C12N15/113C12N15/85C12N2310/113C12N2510/00C12N2800/107
Inventor 裴雪涛曲洺逸谢小燕岳文曾泉房芳陈琳南雪姚海雷
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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