Specific targeted hDGK theta gene luciferase reporting system

Abstract

The invention discloses a specific targeted hDGK theta gene luciferase reporting system. The specific targeted hDGK theta gene luciferase reporting system is composed of a targeting carrier and a targeting donor, wherein an exogenous DNA fragment required to be introduced is carried by the targeting donor, the exogenous DNA fragment comprises T2A small peptide, a luciferase gene cDNA sequence, aneGFP expression frame, a Neomycin gene sequence, and an upstream and downstream homologous arm sequence; and the targeting carrier contains a nuclease Cas9 expression frame and a guiding chain sgRNA expression frame of the targeted hDGK theta. An establishing method of the reporting system comprises the following steps: screening sgRNA of a targeted hDGK theta gene 3' non-coding region, constructing an eukaryotic expression carrier carried by Cas9 and a sgRNA expression element as the targeting carrier, constructing the upstream and downstream homologous arms carried with a luciferase gene cDNA sequence and used for targeted integration as well as the targeting donor used for gene screening, performing co-transfection of target cells by two carriers, screening by using a resistant gene, and establishing the luciferase reporting system of targeted hDGK theta in a target cell.

Description

[0001] The invention belongs to the fields of biotechnology and pharmacology, and relates to a luciferase reporter system, in particular to a luciferase reporter system specifically targeting hDGKθ gene, which can be used for expression of endogenous human diacylglycerol kinase (hDGKθ) Activity monitoring can also be used to screen new drugs that regulate DGKθ. Background technique

[0002] Diacylglycerol Kinases (DGKs) are important endogenous lipid-regulating enzymes, which can simultaneously regulate the concentration of two second messengers in cells—DAG and PA, and participate in various signaling pathways in cells. DGKθ is the only isoform of DGKs isoenzyme type V, and it is also one of the least understood DGK subtypes. DGKθ was originally discovered in mouse brain tissue and structurally contains three cysteine-rich domains (CRD), which distinguish it from other isoforms (containing two CRD domains). In addition, its N-terminus has a proline / glycine-rich domain, pleck...

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