Generation of a novel HEK293 luciferase reporter cell line by CRISPR/Cas9-mediated site-specific integration in the genome to explore the transcriptional regulation of the PGRN gene

Bioengineered. 2019 Dec;10(1):98-107. doi: 10.1080/21655979.2019.1607126.

Abstract

Progranulin has multiple functions in several physiological and pathological processes, including embryonic development, wound repair, tumorigenesis, inflammation and neurodegeneration. To investigate the transcriptional regulation of the PGRN gene, a luciferase knock-in reporter system was established in HEK293 cells by integrating luciferase gene in the genome controlled by the endogenous PGRN promoter using CRISPR/Cas9. PCR results demonstrated the site-specific integration of the exogenous luciferase gene into the genome. To validate the novel luciferase knock-in system, a CRISPR/Cas9 transcription activation/repression system for the PGRN gene was constructed and applied to the knock-in system. In addition, phorbol ester (phorbol 12-myristate, 13-acetate), previously reported as activating the expression of PGRN, was applied to the system. The results indicated that luciferase activity was directly correlated with the activity of the PGRN endogenous promoter. This novel system will be a useful tool for investigating the transcriptional regulation of PGRN, and it has great potential in screening the drugs targeting PGRN.

Keywords: CRISPR/Cas9; HEK293; PGRN; knock-in; luciferase; transcriptional regulation.

MeSH terms

  • Base Sequence
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Founder Effect*
  • Gene Editing
  • Gene Expression Regulation / drug effects
  • Gene Knock-In Techniques / methods*
  • Genes, Reporter
  • Genome, Human
  • HEK293 Cells
  • Humans
  • Luciferases / genetics*
  • Luciferases / metabolism
  • Progranulins / agonists
  • Progranulins / genetics*
  • Progranulins / metabolism
  • Promoter Regions, Genetic / drug effects
  • Signal Transduction
  • Tetradecanoylphorbol Acetate / pharmacology
  • Transcription, Genetic / drug effects

Substances

  • GRN protein, human
  • Progranulins
  • Luciferases
  • CRISPR-Associated Protein 9
  • Tetradecanoylphorbol Acetate

Grant support

This work was supported by research grants to H.X. from the National Natural Science Foundation of China [No. 81471772 and No. 81773265] and the Key Research and Development Plan of Shaanxi Province [No. 2018SF-106].