Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Construction and application of cell model capable of stably expressing human MATE1 transporter

A cell model and stable expression technology, applied to cells modified by introducing foreign genetic material, recombinant DNA technology, microbial measurement/testing, etc., can solve the problems of reduced expression, difficult source of primary cells, low expression, etc.

Inactive Publication Date: 2014-12-17
ZHEJIANG UNIV
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many commonly used human cell lines have low expression or no expression of transporters; the source of primary cells is difficult, and the expression level of transporters on primary cells will also decrease with the extension of in vitro culture time

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Construction and application of cell model capable of stably expressing human MATE1 transporter
  • Construction and application of cell model capable of stably expressing human MATE1 transporter
  • Construction and application of cell model capable of stably expressing human MATE1 transporter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of pcDNA3-hMATE1 recombinant plasmid

[0031] 1. Obtaining the coding sequence of SLC47A1 gene

[0032](1) Design two PCR primers according to the coding sequence of human SLC47A1 gene mRNA, and add a restriction site Hind at both ends of the primers according to the amplification vector pMD19-T and the expression vector pcDNA3.1(+)-hgromycin For the sequences of III and Kpn I (restriction sites are underlined), refer to the sequences of primers No. 1 and 2 in Table 1, and the primers were prepared at a concentration of 10 μmol / L.

[0033] Primer sequences used in the present invention in table 1

[0034]

[0035] (2) TRIzol method Total RNA was extracted from frozen human kidney tissue, and a cDNA library was obtained by reverse transcription PCR. The reverse transcription PCR reaction system is shown in Table 2.

[0036] Table 2 Reverse transcription reaction system (10μL)

[0037] Reagent name Theoretical usage Actual added ...

Embodiment 2

[0059] Example 2 Screening to obtain the MDCK-hMATE1 cell model overexpressing hMATE1

[0060] 1. Transfection of pcDNA3.1(+)-hMATE1 recombinant plasmid into MDCK cells

[0061] (1) Plant MDCK cells in a 6-well plate at a certain density, and when the cells grow to a confluence of 80%, transfect pcDNA3.1(+)-hMATE1 recombinant plasmid and transfection reagent at 10 μg recombinant plasmid: 8 μL After mixing the ratio of the reagents, add 100 μL of DMEM high-glucose medium, and then add dropwise to the MDCK cell culture wells cultured in DMEM medium without FBS. After 6 hours, replace with DMEM medium containing FBS to continue the culture.

[0062] (2) After 48 hours of transfection, replace with DMEM medium containing 400 μg / ml hygromycin and 10% FBS for culture, and change the medium every day or every other day depending on the number of dead cells, and continue to screen for 12 to 14 days.

[0063] (3) When the cells to be screened have no obvious death and are in a good gr...

Embodiment 3

[0088] Example 3 Screening to obtain MDCK-hOCT1 / hMATE1 and MDCK-hOCT2 / hMATE1 cell models overexpressing hMATE1

[0089] 1. The method steps of screening monoclonal are the same as the experimental method of MDCK-hMATE1 monoclonal screening. see attached results Figure 9 , 10 , 12, 13.

[0090] 2. Use the selected MDCK-hOCT1 / hMATE1 and MDCK-hOCT2 / hMATE1 cell models

[0091] The relative content of hMATE1 and hOCT1 / hOCT2 mRNA was determined by Real-Time PCR.

[0092] The cell culture method, the total RNA extraction method steps and reagents, the reverse transcription system conditions and the Real-Time PCR method are all the same as those in item 5 of Example 2, and the primers used for Real-Time PCR are shown in Table 1. SEQ ID No: 3~ 10, see the result Figure 11 , 14 . Figure 11 Among them, the cells transfected with the recombinant plasmid pcDNA3.1-hMATE1 expressed hMATE1 higher than the cells transfected with no load, and the expression of hOCT1 in the double-tran...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a method for constructing a pcDNA3.1(+)-hMATE1 recombinant plasmid. The pcDNA3.1(+)-hMATE1 recombinant plasmid is used for constructing three cell models: MDCK-hMATE1, MDCK-hOCT1 / hMATE1 and MDCK-hOCT2 / hMATE1. Stably high expression of hMATE1 on MDCK cells can be carried out with the constructed MDCK-hMATE1 model. The MDCK-hMATE1 model can be used as a research model for screening a substrate and an inhibitor of the hMATE1 at a high speed and a high flux, can predict a drug-drug interaction induced by the hMATE1. A double-transfection cell model of the MDCK-hOCT1 / hMATE1 and the MDCK-hOCT2 / hMATE1 in the invention can be used for researching transfer of a drug of common substrates of the hOCT1 / hOCT2 and the hMATE1.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to the construction of a Madin-Daby canine kidney epithelial (Madin-Daby canine kidney cells, MDCK) cell model stably expressing human multidrug and toxin excretion protein 1 (hMATE1) transporter, and co-transfecting human organic cations Construction of MDCK cell model of transporter 1 (human organic cation transporter 1, hOCT1) and hMATE1, construction of MDCK cell model of co-transfection of human organic cation transporter 2 (human organic cation transporter 2, hOCT2) and hMATE1, and three Applications of cell models. technical background [0002] With the in-depth study of drug metabolism, the role of zero-phase metabolism and three-phase metabolism mediated by drug transporters in drug disposition in vivo has attracted more and more attention. So far, various transporters of the solute carrier (SLC) superfamily and ATP-binding carrier (ABC) superfamily have been discovered,...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N5/10C12Q1/02
Inventor 雷红梅孙思源李丽萍涂美娟王凯白梦如周慧曾苏蒋惠娣
Owner ZHEJIANG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com