Document Type
Article
Publication Date
3-5-2026
Original Citation
Uenaka T,
Napole A,
Saha A,
Sun D,
Singavarapu A,
Calzada E,
Chen J,
Erlebach L,
McQuade A,
Ramos D,
Rigamonti A,
Salazar L,
Samelson A,
Sedov K,
Welsh N,
Wild K,
Wu Q,
Arenas E,
Bassett A,
Kampmann M,
Kronenberg-Versteeg D,
Merkle F,
Schüle B,
Thompson L,
Skarnes W,
Ward M,
Wernig M.
Prevention of transgene silencing during human pluripotent stem cell differentiation. Cell Stem Cell. 2026;33(3):517-30 e8.
Keywords
JGM, Humans, Cell Differentiation, Transgenes, Pluripotent Stem Cells, Gene Silencing, Promoter Regions, Genetic, Genetic Vectors, Neurons
JAX Source
Cell Stem Cell. 2026;33(3):517-30 e8.
ISSN
1875-9777
PMID
41690310
DOI
https://doi.org/10.1016/j.stem.2026.01.007
Abstract
Transgenes are often silenced upon differentiation of pluripotent stem cells using conventional expression systems. Here, we developed the TK4 PiggyBac vector to conduct a comparative analysis to evaluate the impact of various promoters, transcriptional regulatory elements, insulators, and genomic integration sites on transgene silencing during neuronal differentiation. Our findings reveal that specific combinations of CAG and Ubc promoters with the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) can prevent transgene silencing during differentiation, whereas chromatin insulators have less impact on sustained expression. Three novel safe harbor loci, distant from known genes, as well as the citrate lyase beta-like (CLYBL) locus, similarly support the prevention of transgene silencing. Remarkably, the TK4 vector showed complete resistance to silencing across various neuronal and microglial differentiation protocols, as independently confirmed by seven laboratories. This construct will be highly useful for assays requiring stable transgene expression during differentiation and holds potential for broad applications in various research fields.
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License