Construction and validation of over-expression of RE1-silencing transcription factor (REST) using PiggyBac transposon inducible vector system in HEK293FT cells

Authors

  • Norhazlin Jusoh (1) Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; (2) Faculty of Applied Sciences, Universiti Teknologi MARA, Perak Branch, Tapah Campus, 35400 Tapah Road, Perak, Malaysia.
  • Norshariza Nordin (1) Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; (3) Malaysian Research Institute on Ageing (MyAgeing®), Universiti Putra Malaysia, Selangor, Malaysia.
  • Nurul Ain Nasim Mohd Yusof (4) Pluripotent Stem Cell Laboratory, Haematology Unit, Cancer Research Centre, Institute for Medical Research (IMR), Block C, National Institute of Health (NIH) Malaysia, Setia Alam, Malaysia.
  • Pike-See Cheah (3) Malaysian Research Institute on Ageing (MyAgeing®), Universiti Putra Malaysia, Selangor, Malaysia; (5) Department of Human Anatomy, Faculty of Medicine and Health Sciences, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
  • King-Hwa Ling (1) Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; (3) Malaysian Research Institute on Ageing (MyAgeing®), Universiti Putra Malaysia, Selangor, Malaysia.

DOI:

https://doi.org/10.31117/neuroscirn.v9i2.509

Keywords:

PiggyBac, RE1-Silencing Transcription Factor (REST), Molecular cloning, Transfection

Abstract

RE1-silencing transcription factor (REST), a key regulator of neural gene expression, modulates ion channel function, neurotransmitter receptor activity, and synaptic plasticity, and its dysregulation has been implicated in neurodegenerative diseases. However, stable overexpression of REST in mammalian cells remains technically challenging, highlighting the need for an efficient and controllable delivery platform. Here, we constructed and validated a PiggyBac-based inducible vector system for regulatable REST overexpression in HEK293FT cells as a proof of concept. The REST-FLAG-P2A-GFP construct was assembled using NEBuilder HiFi DNA Assembly and validated by restriction fragment length polymorphism (RFLP), Sanger sequencing, and whole-plasmid sequencing. HEK293FT cells were transfected with REST-FLAG-P2A-GFP and SB100X, together with Xlone_GFP plasmids, using polyethylenimine (PEI), followed by doxycycline induction at 24 and 48 hours post-transfection. REST expression was confirmed by eGFP fluorescence imaging, while blasticidin resistance supported stable transgene integration for up to 5 days. Western blot analysis further verified inducible REST overexpression, detecting REST protein at approximately 130 and 200 kDa, with 1.45-fold (p < 0.01) and 1.56-fold (p < 0.05) increases, respectively, compared with uninduced cells. Collectively, these findings demonstrate the utility of the PiggyBac transposon system for stable and inducible expression of transcription factors in mammalian cells and establish a platform for future studies of REST function and gene regulatory mechanisms.

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2026-06-30

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Jusoh, N., Nordin, N., Mohd Yusof, N. A. N., Cheah, P.-S., & Ling, K.-H. (2026). Construction and validation of over-expression of RE1-silencing transcription factor (REST) using PiggyBac transposon inducible vector system in HEK293FT cells. Neuroscience Research Notes, 9(2), 509.1–509.16. https://doi.org/10.31117/neuroscirn.v9i2.509

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Vol. 9 No. 2 (2026): Regular Issue

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Technical Notes

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Copyright (c) 2026 Norhazlin Jusoh, Norshariza Nordin, Nurul Ain Nasim Mohd Yusof, Pike-See Cheah, King-Hwa Ling

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