TY  - JOUR
AU  - Uenaka, Takeshi
AU  - Napole, Alan
AU  - Saha, Aninda Dibya
AU  - Sun, Duo
AU  - Singavarapu, Angelina
AU  - Calzada, Elizabeth
AU  - Chen, Jiahui
AU  - Erlebach, Lena
AU  - McQuade, Amanda
AU  - Ramos, Daniel M
AU  - Rigamonti, Alessandra
AU  - Salazar, Lisa
AU  - Samelson, Avi J
AU  - Sedov, Kamilla
AU  - Welsh, Natalie J
AU  - Wild, Katleen
AU  - Wu, Qianxin
AU  - Arenas, Ernest
AU  - Bassett, Andrew R
AU  - Kampmann, Martin
AU  - Kronenberg-Versteeg, Deborah
AU  - Merkle, Florian T
AU  - Schüle, Birgitt
AU  - Thompson, Leslie M
AU  - Skarnes, William C
AU  - Ward, Michael E
AU  - Wernig, Marius
TI  - Prevention of transgene silencing during human pluripotent stem cell differentiation.
JO  - Cell stem cell
VL  - 33
IS  - 3
SN  - 1934-5909
CY  - Amsterdam [u.a.]
PB  - Elsevier
M1  - DZNE-2026-00259
SP  - 517 - 530.e8
PY  - 2026
AB  - 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.
KW  - Humans
KW  - Cell Differentiation: genetics
KW  - Transgenes: genetics
KW  - Pluripotent Stem Cells: cytology
KW  - Pluripotent Stem Cells: metabolism
KW  - Gene Silencing
KW  - Promoter Regions, Genetic: genetics
KW  - Genetic Vectors: genetics
KW  - Neurons: cytology
KW  - Neurons: metabolism
KW  - PiggyBac vector (Other)
KW  - UCOE (Other)
KW  - WPRE (Other)
KW  - chromatin insulator (Other)
KW  - human pluripotent stem cells (Other)
KW  - microglia (Other)
KW  - neuron (Other)
KW  - promoter activity (Other)
KW  - safe harbor locus (Other)
KW  - transgene silencing (Other)
KW  - ubiquitous chromatin opening element (Other)
KW  - woodchuck hepatitis posttranscriptional regulatory element (Other)
LB  - PUB:(DE-HGF)16
C6  - pmid:41690310
DO  - DOI:10.1016/j.stem.2026.01.007
UR  - https://pub.dzne.de/record/285483
ER  -