Caco-2 cell model for CRISPR/CAS9-mediated drug transporter targeted knockout and method thereof

A cell model and transporter technology, applied in the field of genetic engineering, can solve problems such as affecting the analysis of results, and achieve the effect of eliminating mutual interference and sensitive cell models

Pending Publication Date: 2018-03-06
SOUTH CHINA UNIV OF TECH
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  • Abstract
  • Description
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Problems solved by technology

However, the cross-reactivity of the three substrates and inhibitors seriously affects the result analysis

Method used

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  • Caco-2 cell model for CRISPR/CAS9-mediated drug transporter targeted knockout and method thereof
  • Caco-2 cell model for CRISPR/CAS9-mediated drug transporter targeted knockout and method thereof
  • Caco-2 cell model for CRISPR/CAS9-mediated drug transporter targeted knockout and method thereof

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Embodiment 1

[0027] In the embodiment of the present invention, the CRISPR / CAS9-mediated drug transporter-targeted knockout method of the caco-2 cell model, which is used for non-diagnostic or therapeutic purposes, specifically includes the following steps:

[0028] 1. Design target-specific sgRNAs for the main members of the ABC family, P-gp, BCRP and MRP2, construct their expression vectors, and test the effectiveness of gene knockout.

[0029] Design specific sgRNAs for the gene expression functional regions of P-gp (NG_011513.1-ABCB1-Pgp), BCRP (NG_032067.2-ABCG2-BCRP) and MRP2 (NG_011798.1-ABCC2-MRP2), and perform off-target analyze. Two sgRNAs with good specificity and low off-target were screened for each gene, and the design results of sgRNAs for P-gp, BCRP and MRP2 are shown in Table 1.

[0030] Table 1

[0031]

[0032] U6 promoter was used to express sgRNA, and the designed sgRNA sequence was synthesized into Oligo to construct sgRNA expression vector U6-sgRNA. All were su...

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Abstract

The invention discloses a caco-2 cell model for CRISPR / CAS9-mediated drug transporter targeted knockout and a method thereof. The method is used for non-diagnostic or therapeutic purposes and comprises the following steps: designing sgRNA with target specificity for P-gp, BCRP and MRP2 transporters and constructing a sgRNA expression vector, wherein a sequence of the designed sgRNA is shown as SEQID NO. 1-6 in a sequence table; respectively designing P-gp, BCRP and MRP gene single knockout and pairwise combination double knockout by utilizing CRISPR / CAS9, co-transfecting a caco-2 cell with anhCas9 plasmid and performing monoclonal expansion culture to obtain the caco-2 cell model for transporter gene targeting. The caco-2 cell model obtained by the method disclosed by the invention has the beneficial effects that the mutual interference among different transporters is effectively eliminated, and a more specific and more sensitive cell model is provided for drug transport research.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a caco-2 cell model and a method for CRISPR / CAS9-mediated targeted knockout of drug transporters. Background technique [0002] Drug transport research is an important part of the new drug development process. The transporter performs secondary active transport through the transporter, that is, it relies on the potential energy of the high concentration of sodium ions in the extracellular space (the potential energy is provided by the primary active transport), which is equivalent to indirectly consuming the energy of ATP. For example, during the active absorption of glucose by the epithelial cells of the small intestinal mucosa, the transport energy does not come directly from the decomposition of ATP, but depends on the concentration gradient of sodium ions between the epithelial cells (low) and the intestinal fluid (high). When the concentration gradient enters the epitheli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85
CPCC07K14/47C12N5/0693C12N15/85C12N2800/80C12N2810/10
Inventor 黄黎珍钟国瑞谢水林庞土慧姜二岗邹淑香李浩健戴仁科
Owner SOUTH CHINA UNIV OF TECH
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