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A kind of carrier and application thereof of shrna that suppresses mouse gh gene expression

A technology of gene expression and carrier, applied in the field of shRNA carrier, can solve the problem of lack of shRNA, and achieve the effect of high expression efficiency

Active Publication Date: 2018-04-27
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is a lack of a shRNA carrier and its application that effectively inhibits mouse GH gene expression

Method used

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  • A kind of carrier and application thereof of shrna that suppresses mouse gh gene expression
  • A kind of carrier and application thereof of shrna that suppresses mouse gh gene expression
  • A kind of carrier and application thereof of shrna that suppresses mouse gh gene expression

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Design and synthesis of GH shRNA

[0037] (1) Synthesis of oligonucleotides

[0038]The nucleotide sequence according to mouse GH mRNA is the nucleotide sequence shown in SEQ ID NO:9. Four shRNAs and a control shRNA were designed for different sites, TTCAAGAGA was selected as the loop structure in the template to avoid the formation of a termination signal, and the transcription termination sequence of the shRNA adopted the T6 structure. CACC is added to the 5' end of the sense strand template, which is complementary to the sticky end formed after digestion with BbsI; GATC is added to the 5' end of the antisense strand template, which is complementary to the sticky end formed after digestion with BamHI; if the first siRNA A base is not G, then add a G after CACC, the sequence names are GH-mus-340, GH-mus-544, GH-mus-616, GH-mus-627, and design a negative control shRNA, so The nucleotide sequence of the sense strand of the negative control is the nucleotide sequence sh...

Embodiment 2

[0067] GH expression protein detection after shRNA interference

[0068] The prepared overexpression vector pGPU6 / GFP / Neo-shRNA containing different GH shRNAs was transfected into the mouse pituitary tumor cell line AtT-20 cells, G418 was screened for 12 days to enrich the cells with integrated exogenous genes, and the total cell protein was extracted , to detect the expression of GH protein in each group of cells.

[0069] Such as Figure 4 with Figure 5As shown, NC in the figure represents the negative control, that is, the group of cells transfected with pGPU6 / GFP / Neo linearized fragment, GH340 represents the group of cells transfected with pGPU6 / GFP / Neo-GH340, and GH544 represents the group of cells transfected with pGPU6 / GFP / Neo-GH544 GH616 represents the group of transfected pGPU6 / GFP / Neo-GH616 cells, and GH627 represents the group of transfected pGPU6 / GFP / Neo-GH627 cells. The test results showed that the relative gray value of negative control NC was 94, transfected...

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Abstract

The invention discloses a shRNA carrier for inhibiting mouse GH gene expression, characterized in that: the shRNA includes GH-mus-340 and GH-mus-340 and GH-544 sites that are specifically combined with the GH mRNA sequence at the 340 position respectively. mus-544, GH-mus-616 at positions 616 and GH-mus-627 at positions 627; the GH-mus-340, GH-mus-544, GH-mus-616, GH-mus-627 The DNA template includes the nucleotide sequence of the siRNA sense strand template, the nucleotide sequence of the loop loop, the nucleotide sequence of the siRNA antisense strand template and the nucleotide sequence of the termination signal. The invention can effectively inhibit mouse GH gene expression, and all four GH shRNA overexpression vectors have high expression efficiency.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an shRNA carrier for inhibiting mouse GH gene expression and application thereof. Background technique [0002] Small interfering RNA siRNA is a small RNA molecule with a nucleotide sequence length of ~21-25, which is processed by Dicer, an enzyme specific to double-stranded RNA in the RNAase III family. SiRNA is the main member of siRISC, which stimulates the silencing of its complementary target mRNA. RNA interference RNA interference, RNAi refers to the specific degradation of intracellular mRNA mediated by endogenous or exogenous double-stranded RNA, dsRNA, resulting in the silencing of the expression of target genes and the loss of corresponding functional phenotypes. [0003] Mouse growth hormone mouse growth hormone, mGH is a single-chain polypeptide hormone secreted by the anterior lobe of the mouse pituitary gland. It can accelerate protein synthesis and lipid metabolism, s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63A61K48/00
Inventor 鞠辉明
Owner YANGZHOU UNIV