Journal Article

DZNE-2026-00259

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Prevention of transgene silencing during human pluripotent stem cell differentiation.

Uenaka, T. ; Napole, A. ; Saha, A. D. ; Sun, D. ; Singavarapu, A. ; Calzada, E. ; Chen, J. ; Erlebach, L.DZNE* ; McQuade, A. ; Ramos, D. M. ; Rigamonti, A. ; Salazar, L. ; Samelson, A. J. ; Sedov, K. ; Welsh, N. J. ; Wild, K.DZNE* ; Wu, Q. ; Arenas, E. ; Bassett, A. R. ; Kampmann, M. ; Kronenberg-Versteeg, D.DZNE* ; Merkle, F. T. ; Schüle, B. ; Thompson, L. M. ; Skarnes, W. C. ; Ward, M. E. ; Wernig, M.

2026
Elsevier Amsterdam [u.a.]

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Please use a persistent id in citations: doi: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.

Keyword(s): Humans (MeSH) ; Cell Differentiation: genetics (MeSH) ; Transgenes: genetics (MeSH) ; Pluripotent Stem Cells: cytology (MeSH) ; Pluripotent Stem Cells: metabolism (MeSH) ; Gene Silencing (MeSH) ; Promoter Regions, Genetic: genetics (MeSH) ; Genetic Vectors: genetics (MeSH) ; Neurons: cytology (MeSH) ; Neurons: metabolism (MeSH) ; PiggyBac vector ; UCOE ; WPRE ; chromatin insulator ; human pluripotent stem cells ; microglia ; neuron ; promoter activity ; safe harbor locus ; transgene silencing ; ubiquitous chromatin opening element ; woodchuck hepatitis posttranscriptional regulatory element

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Contributing Institute(s):

1. Cell Biology of Neurological Diseases (AG Jucker)
2. Neuroimmunology and Neurodegenerative Disease (AG Neher (Tübingen))
3. Glial Cell Biology (AG Kronenberg-Versteeg)

Research Program(s):

1. 352 - Disease Mechanisms (POF4-352) (POF4-352)
2. 351 - Brain Function (POF4-351) (POF4-351)

Appears in the scientific report 2026

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Database coverage:
; BIOSIS Previews ; Biological Abstracts ; Clarivate Analytics Master Journal List ; Current Contents - Life Sciences ; Essential Science Indicators ; IF >= 20 ; JCR ; SCOPUS ; Science Citation Index Expanded ; Web of Science Core Collection

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