Specific targeted hDGK theta gene luciferase reporting system

A luciferase and reporter system technology, which is applied in the luciferase reporter system specifically targeting the hDGKθ gene, the luciferase reporter system, and the field of new drug screening. Source DGKθ reporting system and other issues

Active Publication Date: 2018-08-03
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the data search conducted by the applicant, so far, no relevant reports on targeting the endogenous DGKθ reporter system have been found, and there are no literature on DGKθ transcriptional regulation and new drug screening for reference.

Method used

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  • Specific targeted hDGK theta gene luciferase reporting system
  • Specific targeted hDGK theta gene luciferase reporting system
  • Specific targeted hDGK theta gene luciferase reporting system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: Establishment of a reporter system targeting DGKθ in HEK293 cells

[0072] 1. The structure of the reporting system targeting DGKθ in the HEK293 cell line is as follows:

[0073] Based on human chromosome 4, the NCBI number of DGKθ genome sequence is NC_000004. On one of the DNA strands, the DNA sequence between 12918bp and 13656bp from the 5' end of the DGKθ genome was removed, and the T2A sequence, luciferase gene sequence, eGFP expression box, T2A sequence, and Neomycin gene sequence were sequentially inserted between 12918bp and 13656bp . On the other allelic chain, a 172bp fragment was randomly inserted at 13171bp from the 5' end of the DGKθ genome.

[0074] 2. The construction process of the reporter system targeting hDGKθ in HEK293 cells is as follows:

[0075] (1) Construction of targeting vector pCas9 / hDGKθsgRNA

[0076] The following primers were synthesized in Huada Gene Company, and the sequences of each primer are as follows:

[0077] P1(hDG...

Embodiment 2

[0109] The following is a specific example of establishing a reporter system targeting DGKθ in HepG2 cells as an example.

[0110] 1. The structure of the reporter system targeting DGKθ in the HepG2 cell line is as follows:

[0111] Based on human chromosome 4, the NCBI number of DGKθ genome sequence is NC_000004. On one of the DNA strands, the DNA sequence between 12918bp and 13656bp from the 5' end of the DGKθ genome was removed, and the T2A sequence, luciferase gene sequence, eGFP expression box, T2A sequence, and Neomycin gene sequence were sequentially inserted between 12918bp and 13656bp . On the other allelic strand, random repair did not change the base composition of the genome.

[0112] 2. The construction process of the reporter system targeting hDGKθ in HepG2 cells (HepG2-hDGKθ-T2A-luciferase-KI cell line) is as follows:

[0113] The construction process is the same as that of the HEK293 cells mentioned in Example 1. attached Figure 5 (A) shows the expression...

Embodiment 3

[0115] The following is the application of the reporter system targeting hDGKθ established in HEK293 cells in drug screening:

[0116] Palmitic acid (PA), oleic acid (OA) and EGCG (Sigma) were purchased for drug testing. Palmitic acid (PA) and oleic acid (OA) were formulated as free fatty acids (FFA) in a ratio of 2:1. The reported hDGKθ inhibitor R59949 was used as the positive control group.

[0117] Will 1×10 5hDGKθ-T2A-Luciferase-HEK293 cells were plated on 24-well cell culture plates, and after 24 hours, drugs were added to induce them. The doses of each group are as follows: FFA (0.5 μM / L) group, EGCG (5 μM / L), R59949 (15 μM / L). DMSO was the negative control group. After 72 hours, the cells in each well were collected and the luciferase activity was detected. attached Image 6 (A) shows changes in luciferase activity induced by drugs. Similar to R59949, FFA decreased luciferase activity, while EGCG enhanced luciferase activity.

[0118] Will 2×10 5 HEK293 cells ...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N9/22
CPCC12N9/22C12N15/907C12N2800/107C12N2810/00
Inventor 夏海滨单琳琳张伟锋赵俊丽
Owner SHAANXI NORMAL UNIV
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