Construction method of primate miRNA-122 knockout model, primate liver cancer model and application thereof

A technology of miRNA-122 and primates, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of unsuitable liver cancer drug screening, unsuitable for human liver cancer mechanism research, etc.

Inactive Publication Date: 2016-06-01
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

As a result, the mouse liver cancer model is not suitable for the study of human liver cancer mechanism, nor is it suitable for the screening of liver cancer drugs. Only primates can be used as effective animal models of liver cancer

Method used

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  • Construction method of primate miRNA-122 knockout model, primate liver cancer model and application thereof
  • Construction method of primate miRNA-122 knockout model, primate liver cancer model and application thereof
  • Construction method of primate miRNA-122 knockout model, primate liver cancer model and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Refer to attached figure 1 . miR-122 directly targets TGFβ1 in human cells and TGFβR1 in mouse cells.

[0069] (a) Western blot analysis of TGFβ1 and TGFβR1 transfected with miR-122 overexpression plasmid (122), miR-122 sponge (122sp) and NC in HepG2, Huh7 and Hepa1-6 cells.

[0070] (b) Western blotting of HepG2 cells transfected with miR-122, NC and miR-122 and TGFβ1, which are respectively intracellular TGFβ1, Smad2 and p-Smad2, and the quantitative analysis results are on the right side of the figure, n=3.

[0071] (c) Western blot of NIT-1 cells transfected with miR-122, NC, and miR-122 and TGFβR1, which are intracellular TGFβR1, Smad2 and p-Smad2, respectively. Quantitative analysis results are on the right side of the figure, n=3.

[0072] (d) Luciferase activity was measured after transfecting the corresponding vector. The corresponding TGFβ1 3'UTR luciferase vector was constructed using the pGL plasmid. Hum: Human; Hum1: long 3'UTR; Hum2: short 3'UTR; Rhe...

Embodiment 2

[0077] Refer to attached figure 2 . miR-122 targets TGFβ15'UTR in a non-canonical "seed region" base-pairing manner.

[0078] (a) Luciferase activity was measured after transfection of corresponding vectors. TGFβ15'UTR was ligated to the promoter region of pGL plasmid. n=6.

[0079] (b) Determination of luciferase activity after transfection of corresponding vectors. The human TGFβ1 5'UTR was truncated into 7 different segments and each segment was ligated into the promoter region of the pGL plasmid. n=6.

[0080] (c) Schematic representation of miR-122 acting on TGFβ15'UTR and mutant regions. The luciferase activities of different vectors are as follows.

[0081] (d) Evolutionary trajectory of miR-122 at TGFβ15'UTR target positions in different animals. Acquisition of the miR-122 target occurred in the common ancestor of manatees with humans and other primates (black arrows), in pigs, dogs, rats, and mice due to several insertions between positions 11 and 12 bases r...

Embodiment 3

[0086] Refer to attached image 3 . Because targeting TGFβ1 or TGFβR1, respectively, resulted in differential effects of miR-122 on mesenchymal transition (EMT) in human and mouse cells.

[0087] (a) Western blot analysis of cadherin and vimentin in Huh7 cells after corresponding treatments. HepG2 and HepG2-122 represent the culture supernatant of HepG2 and HepG2-122 cells, respectively. Quantitative analysis results are on the right side of the figure, n=3.

[0088] (b) Nuclei were stained with DAPI using cadherin and vimentin as EMT labels in Huh7 cells. Scale bar: 50 μm.

[0089] (c) Western blot analysis of cadherin and vimentin in Huh7 cells after corresponding treatments. Hepa1-6 and Hepa1-6-122sp represent the culture supernatant of Hepa1-6 and Hepa1-6-122sp cells, respectively. Quantitative analysis results are on the right side of the figure, n=3.

[0090] (d) Nuclei were stained with DAPI using cadherin and vimentin as EMT labels for Huh7 cells. Scale bar: 50...

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Abstract

A construction method of a primate liver cancer model comprises the following steps: a) inserting gRNA sequence for specifically targeting miR122 into an appropriate vector to construct a template vector for in vitro transcription of miR122; b) inserting cas9 gene sequence into an appropriate vector to construct a template vector for in vitro transcription of cas9 gene; c) carrying out PCR amplification by using the vector in the Step a) so as to obtain a gRNA temperate for in vitro transcription of miR122; d) linearizing the template vector in the Step b) by the use of restriction enzyme so as to obtain a template for in vitro transcription of cas9 gene; e) carrying out in vitro transcription by using products in the Step c) and Step d) as templates so as to obtain gRNA for specifically targeting miR122 and mRNA of cas9 gene; f) injecting the gRNA and mRNA in the Step e) into fertilized egg cell of the animal so as to generate stable miR122-knockout animal offspring.

Description

technical field [0001] The invention relates to a method for constructing a primate liver cancer model, an animal liver cancer model and a method for screening drugs with the model. In particular, it relates to a method for constructing a monkey liver cancer model with miRNA miR-122 expressed in the liver. Background technique [0002] Hepatocellular carcinoma is the most common liver tumor and ranks third among cancer-related deaths worldwide, with more than 500,000 new patients worldwide each year. More than 28,700 new cases are expected in the United States in 2012, and 20,550 are expected to die from this malignancy. Major risk factors for HCC include hepatitis B and C virus infection and liver damage from alcohol consumption. If discovered in time, there is hope for a cure, but most cases are at an advanced stage when they are diagnosed. [0003] In 2012, researchers from the Ohio State University Comprehensive Cancer Center confirmed that a small molecule called mic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/873A01K67/027A61K49/00
Inventor 席建忠孙常宏
Owner PEKING UNIV
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