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Nucleic Acids and Their Uses

A technology of nucleic acid and nucleotide sequence, applied in the direction of nucleic acid carrier, the use of carrier to introduce foreign genetic material, genetically modified cells, etc., to reduce the difficulty of synthesis and increase the effect of down-regulating miRNA

Active Publication Date: 2019-12-20
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 Acids and Their Uses
  • Nucleic Acids and Their Uses
  • Nucleic Acids and Their Uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0036] After the functionally down-regulated miRNA is clearly required, the binding sequence of the target gene that has been reported or can be predicted can be queried, and the binding sequence of the target gene is connected in series.

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

[0038] Table 1:

[0039]

[0040] The designed sequence capable of functionally down-regulating miR-1915-3p is as follows:

[0041]

[0042] The underlined sequence is the miRNA target gene binding site sequence, the bold underlined part is the complementary pairing sequence of the miRNA target gene, and 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 by EcoRI and EcoRⅤ ( figure 1 The agarose gel electrophoresis pattern of the fragment synthesized in Example 1 with EcoRI and EcoRⅤ digestion), and it is ligated into the purpose plasmid (plasmid pattern is as follows: figure 1 shown).

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

[0047] Table 2:

[0048] Composition quantity plasmid 4μg EcoRI 10IU EcoRⅤ 10IU 10×Buffer 4μl water Add to 40μl

[0049] Mix the components of the enzyme 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 puri...

Embodiment 3

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

[0057] 1. Establishment of cell lines stably down-regulating miR-1915-3p expression

[0058] (1) Human leukemia cells (such as K562 cells or UT-7 cells) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), and passaged at a ratio of 1:5 every 3 days. Count the leukemia cell line after centrifugation, and resuspend in culture medium to a cell density of 3×10 5 pcs / ml, add 1.5ml per well into the marked 6-well plate;

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

[0060] (3) Mix the liquids in EP tubes 1 and 2, and mix them gently. After standing at room temperature for 20 minutes, add the liquid corresponding to the cell suspension in the pre...

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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 an isolated nucleic acid, a method for down-regulating miRNA in cells, and a method for establishing a cell line stably down-regulating miRNA. Background technique [0002] microRNA (miRNA) is a class of single-stranded non-coding RNA produced by endogenous expression in eukaryotes. miRNA can bind to the 3'UTR region of the mRNA transcribed by the target gene in the form of complementary base pairing, and very few bind to the 5'UTR region or the ORF region, degrade the mRNA of the target gene, or inhibit its translation, thereby down-regulating the target protein. 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, inflammatory response and other physiological and case conditions. important role. However...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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