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
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  • 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

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  • 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...

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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

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Application Information

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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
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