In-vitro evaluation cell model of skin sensitization of compound and construction method of cell model

Abstract

The invention discloses an in-vitro evaluation cell model of skin sensitization of a compound and a construction method of the cell model. The construction method of the cell model comprises the following steps of: designing and constructing an sgRNA (small guide ribonucleic acid) expression vector with CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9), designing and constructing a homologous recombinant vector capable of knocking a reporter gene connected with a spontaneous lysis peptide sequence into an expression cassette of an HMOX1 (heme oxygenase (decycling) 1) gene, and cotransfecting a cell with the homologous recombinant vector, hCas9 (humanized Cas9) plasmid and the sgRNA expression vector for monoclone enlarging culture to form the cell model. The HaCaT cell model with a luciferase gene knocked in before a termination codon of the HMOX1 gene by a combined CRISPR / CAS9 cell monoclone technology. The cell model achieves synchronous expression of the luciferase gene and the HMOX1 gene, so that a sensitization compound and a non-sensitization compound are effectively differentiated, and the more specific and sensitive cell model is provided for compound sensitization studies.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a compound skin sensitization evaluation cell model in vitro and its construction method, in particular to a CRISPR / CAS9-mediated HMOX1 gene targeted knock-in reporter gene cell model and its method. Background technique [0002] Skin sensitization is a delayed hypersensitivity reaction caused by skin contact with elicitors. The sensitizing compound combines with endogenous proteins to form a hapten, which causes the activation of skin dendritic cells and a series of reactions in keratinocytes, which in turn activates T cells, resulting in Lymph node hyperplasia and body skin inflammation, this process is also called skin sensitization harmful outcome pathway AOP. [0003] Animal experiments for skin sensitization mainly include guinea pig skin test and local lymph node assay (LLNA) in mice. Among them, the improved version of tracer LLNA:BrdU ELISA has become the most ...

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Problems solved by technology

[0005] However, these reporter cell models only indirectly reflect target gene expression, rather than real-time reporter gene expression

The essence of the model is a transgenic cell in which the regulatory element-luciferase is randomly inserted. Although the cell can indirectly evaluate the expression of the target gene through the expression of luciferase to a certain extent, it is not a real reporter cell that tracks the expression of the endogenous gene synchronously. As a result, the model can only evaluate whether the compound is sensitized, and it is difficult to quantitatively evaluate the sensitization strength

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