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Biotechnology Letters

, Volume 40, Issue 11–12, pp 1495–1506 | Cite as

Establishment of a HEK293 cell line by CRISPR/Cas9-mediated luciferase knock-in to study transcriptional regulation of the human SREBP1 gene

  • Zihang Li
  • Junli Zhao
  • Niaz Muhammad
  • Dongyang Wang
  • Qinwen Mao
  • Haibin XiaEmail author
Original Research Paper

Abstract

Objectives

To establish a HEK293 cell line with a luciferase knock-in reporter controlled by the endogenous SREBP1 promoter for investigating transcriptional regulation of the SREBP1 gene.

Results

PCR confirmed the site-specific integration of a single copy of the exogenous luciferase gene into one allele of the genome and a 14 bp deletion of the targeted sequence in the other. Luciferase activity was directly correlated with the promoter activity of the endogenous SREBP1 gene in the HEK293-SREBP1-T2A-luciferase-KI cell line cell line.

Conclusions

We successfully generated a novel luciferase knock-in reporter system, which will be very useful for studying transcriptional regulation of the SREBP1 gene and for screening drugs or chemical molecules that regulate SREBP1 gene expression.

Keywords

SREBP1 CRISPR/Cas9 Transcriptional regulation Knock-in Cell line 

Notes

Acknowledgements

This work was supported by the Fundamental Research Funds for the Central Universities, Shaanxi Normal University (No. 2016CSY015), and 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).

Supporting information

Supplemental Fig. 1—Assay of the pGL3-SREBP1p-mCherry vector in HEK293 cells. Control vector pGL3-mCherry and experimental vector pGL3-SREBP1p-mCherry were transfected into HEK293 cells for 48 hours, respectively. Then, mCherry expression was detected under fluorescence microscope in the control vector (A and B) and experimental vector (C and D).

Supplemental Fig. 2—Illustration of the position of the sgRNA target sequence in the genome. The target regions of sgRNA were graphed according to their location, between − 1470 bp and + 1 bp of the SREBP1 transcription start site (TSS).

Supplemental Table 1—Oligonucleotide sequences for SREBP1 sgRNAs.

Supplemental Table 2—PCR primers used in this study.

Supplemental Table 3—Oligonucleotide sequences for SREBP1 promoter sgRNAs.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Laboratory of Gene Therapy, Department of Biochemistry, College of Life SciencesShaanxi Normal UniversityXi’anPeople’s Republic of China
  2. 2.Department of PathologyNorthwestern University Feinberg School of MedicineChicagoUSA

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