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Human oat1-mrp2-ugt2b7 triple stable transfection cell line and its construction method

A technology of OAT1-MRP2-UGT2B7, transfected cells, applied in animal cells, vertebrate cells, urinary tract/kidney cells, etc., to achieve the effect of strong reproducibility, stable transfection method, and efficient construction

Active Publication Date: 2021-03-12
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there is a lack of a OAT1-MRP2-UGT2B7 stably transfected cell line

Method used

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  • Human oat1-mrp2-ugt2b7 triple stable transfection cell line and its construction method
  • Human oat1-mrp2-ugt2b7 triple stable transfection cell line and its construction method
  • Human oat1-mrp2-ugt2b7 triple stable transfection cell line and its construction method

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Experimental program
Comparison scheme
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Embodiment 1

[0029] The construction of embodiment 1 plasmid

[0030] Using synthetic biology methods, hOAT1, hMRP2, and hUGT2B7 target genes were obtained through gene synthesis, wherein the nucleotide sequences of hOAT1, hMRP2, and hUGT2B7 target genes were respectively as SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3 shown. The above three genes were respectively connected to pcDNA3.1 / Hygro(+), pcDNA3.1 / G418(+), pcDNA3.1 / Puromycin(+) expression vectors, after transformation and amplification, the plasmid pcDNA3.1 / Hygro was extracted and purified (+)-hOAT1, pcDNA3.1 / G418(+)-hMRP2, pcDNA3.1 / Puromycin(+)-hUGT2B7 recombinant plasmids. The recombinant plasmid was identified by electrophoresis, and the corresponding target band was obtained. Using Nhe I and Kpn I as the restriction endonuclease site (or Nhe I and Hind III), the recombinant plasmid was identified by double enzyme digestion, and the corresponding target band was obtained. Vector bands and target gene bands, the results show that ...

Embodiment 2

[0031] Transfection of embodiment 2 plasmid

[0032] (1) Source and culture of cells:

[0033] The transfected cells were MDCKII cells, and canine kidney epithelial cells (MDCKII) were purchased from ECACC (European Collection of Cell Lines / Microorganisms). The cells were grown in 100mm cell culture dishes and passaged once every three days at a ratio of 1:50. Humidity, cultured in a 37°C incubator.

[0034] (2) Antibiotic concentration screening:

[0035]Take the MDCKII cells in the logarithmic growth phase to screen the concentration of antibiotics (G418, hygromycin B, Puromycin), the antibiotic concentration ranges from 100μg / mL to 1000μg / mL, culture for 2-3 weeks, and select the lowest antibiotic that kills all the cells The concentration was used as the antibiotic concentration used in the construction of stable transfected cell lines. The results showed that the selection concentration of G418 in MDCKII cells was 800μg / mL, hygromycin B was 400μg / mL, and Puromycin was...

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Abstract

The invention relates to a construction method and functional verification of a human OAT1-MRP2-UGT2B7 triple stable transfection cell strain. The method comprises the following steps: assembling a target gene of hOAT1 with a pcDNA3.1 / Hygo (+) expression vector, assembling a target gene of hMRP2 with a pcDNA3.1 / G418(+) expression vector, assembling a target gene of hUGT2B7 with a pcDNA3.1 / Puromycin (+) expression vector, constructing pcDNA3.1 / Hygro (+)-HOAT1 plasmid, pcDNA3.1 / G418(+)-HMRP2 plasmid and pcDNA3.1 / Puromycin (+)-hUGT2B7 plasmid; wherein the nucleotide sequences of the target genesof hOAT1, hMRP2 and hUGT2B7 are respectively shown as SEQ ID No. 1-3; the three kinds of plasmid constructed by a liposome method are sequentially transfected into MDCKII cells, Caco 2 cells or HEK293cells, specific antibiotics are respectively used for screening positive clones, and positive compounds are used for carrying out transport or metabolic function verification to confirm that the cellstrain stably expresses OAT1, mRP2 and UGT2B7 genes and proteins thereof. The triple stable transfection cell strain can be used for simulating OAT1-mediated uptake in liver, MRP2-mediated efflux andUGT2B7-mediated biphasic metabolic function, and is an important in-vitro research means for evaluating a drug treatment mechanism.

Description

technical field [0001] The invention relates to the field of cell line construction, in particular to a construction method and functional verification of a human OAT1-MRP2-UGT2B7 triple stable transfection cell line. Background technique [0002] Drug metabolism and transport is a key factor in drug disposal in vivo, which directly affects the safety and therapeutic effect of drugs. With the development of biotechnology, the establishment of high-expression cell models in vitro has increasingly become the preferred model for studying drug disposition mechanisms, interactions, and safety. Currently, the reported cell models for the study of metabolic transport include Caco2 cells (human colorectal cancer cell line), high-expression transporter MDCKII cell models (such as MDCKII-hOCTs, MDCKII-hOATPs, MDCKII-hMDR1, MDCKII-hBCRP, MDCKII- MRP2, MDCKII-hMDR1-hBCRP, etc.) and HEK293 cell models (such as HEK293-OATP1A2, HEK293-OATP1B1, HEK293-OCT, etc.). The establishment of thes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10
CPCC12N5/0686C12N15/85
Inventor 张洪建王美玉
Owner SUZHOU UNIV