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Dual-illumination reporting system and application thereof

A reporting system, fluorescent signal technology, applied in the introduction of foreign genetic material using vectors, biochemical equipment and methods, compound screening, etc. Effect

Inactive Publication Date: 2018-08-28
RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The low immunity of tumor patients leads to secondary severe infection and even death; 2. Nutritional depletion and metabolic disorders

Method used

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  • Dual-illumination reporting system and application thereof
  • Dual-illumination reporting system and application thereof
  • Dual-illumination reporting system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Experimental materials and equipment:

[0040] Q5 ultra-fidelity 2X Master Mix (NEB#M0494S), endonucleases Xhol, BamHI, EcoRI, Nhel, Clal, Xbal (all from NEB), T4 ligase (Takara), gDNA extraction kit (AxyPrepTM Blood gDNAMiniprep Kit) , Plasmid Miniprep Kit (AxyPrepTM Plasmid Miniprep Kit), pBlunt-EasyClone vecor (Competent Gold Biotechnology), Dh5α Competent Bacteria (Competent Gold Biotechnology), PC-3 cell line ( CRL-1435 TM ), Ampicillin (Sigma), Gel Extraction Kit (EZNATM Gel Extraction Kit), Transfection Reagent (Cat#114-07); TGF-β (PeproTech, Cat.96-100-21C, 100ng / ml); pLKO.1 (Addgene#8453)

[0041] Experimental steps:

[0042] Step 1. gDNA extraction and plasmid miniprep

[0043] 1) Take 500uL of normal human peripheral blood, extract genomic DNA according to the operation instructions of the gDNA extraction kit, and use it as a template for PCR cloning of Ecad and Vim promoters;

[0044] 2) Prepare liquid LB medium (tryptone 1% w / v, yeast extract 0.5% w / ...

Embodiment 2

[0077] Example 2: Dynamic detection of living cell EMT or MET conversion process

[0078] Experimental materials: the E-V dual fluorescent reporter system obtained in Example 1, TGF-β (PeproTech, Cat.96-100-21C, 100ng / ml), PC3 cell line ( CRL-1435 TM ), flow cytometer (BD FACSAria TM II), cell culture dish, DMEM medium (GibcoTM#C11965500BT), FBS (Gibco TM #10099141), P / S (GibcoTM#10378016), Leica Live Cell Workstation;

[0079] Experimental steps:

[0080] 1) Inoculate the human prostate cancer cell line PC-3 on a 3.5cm glass-bottom culture dish in 10% FBS-DMEM, 5% CO 2 , cultured at 37°C;

[0081] 2) When cultured to a cell density of 50%-60%, refer to Transfection Reagent Instructions Transfect the E-V dual fluorescent reporter system plasmid into PC-3, please refer to the specific transfection method Operating instructions for transfection reagents;

[0082] 3) After 12 hours of transfection with the E-V dual fluorescent reporter system, replace the medium with ...

Embodiment 3

[0086] Example 3: Rapid screening of drugs, compounds and biomacromolecules that can affect cell EMT or MET

[0087] Experimental materials: PC3 cell line, 96-well cell culture plate, E-V double chemiluminescent reporter system described in Example 1, TGFβ, cell culture dish, DMEM medium (GibcoTM#C11965500BT), FBS (GibcoTM#10099141), P / S (GibcoTM#10378016), microplate reader; PC3 cell line ( CRL-1435 TM );

[0088] Experimental steps:

[0089] 1) Seed PC3 cells into 96-well plates in 10% FBS-DMEM, 5% CO 2 , cultured at 37°C;

[0090] 2) When the cells grow to a density of 60%-70%, use Transfer the E-V double chemiluminescent system to the cells, and after continuing to culture for 12 hours, replace the fresh medium, and add TGF-β at a concentration of 100ng / ml and continue to culture;

[0091] 3) After adding TGF-β for 24 hours, suck off the medium, and refer to According to the operation instructions in ReporterAssay (Promega Cat: E1960), add 50uL 1x PLB (included ...

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Abstract

The invention provides a dual-illumination reporting system and application thereof. The dual-illumination reporting system comprises an ECAD promoter fragment and a VIM promoter fragment, and furthercomprises a luciferase gene fragment and a Renilla gene fragment which are connected with the ECAD promoter fragment and the VIM promoter fragment respectively, or further comprises a mCherry gene fragment and an eGFP gene fragment which are connected with the ECAD promoter fragment and the VIM promoter fragment respectively. Molecular targeted medicine screening based on the EMT or the MET can be achieved.

Description

technical field [0001] The invention relates to an epithelial-mesenchymal transition double-luminescent reporter system and its application. Background technique [0002] In the general environment of the entire global society, cancer is a heavy burden in both developed and developing countries. Due to the increasing population, bad living habits such as smoking, drinking, drinking and lack of exercise, and the rapid economic development, the incidence of tumors shows a high growth rate. Based on GLOBOCAN assessment statistics, in 2012, there were 14.1 million new cancer cases and 8.2 million deaths from cancer worldwide. Among them, lung cancer, breast cancer, colon cancer, prostate cancer, etc. can be called the number one killer in tumors. [0003] At present, surgical resection is the main clinical treatment for obvious tumor lesions, while radiotherapy and chemotherapy are the main treatments for tumor cells that are free from blood vessels, have no obvious lesions, a...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65C12Q1/02
CPCC12N15/65C12N15/85C12N2503/02C12N2800/107G01N33/5011G01N33/5044G01N2500/10
Inventor 朱鹤高维强吉忠忠程姹萍
Owner RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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