Takeshi Uenaka

Only smiles in the tissue culture room from now on 🙂
Find the TK4 plasmids from the Wernig lab here:
www.addgene.org/browse/artic...

Have you ever cried in the tissue culture room because your #iPSC transgene got silenced after differentiation?

I have. Many, many times.

So we built TK4, a PiggyBac vector designed to resist silencing.

Now available on @addgene.bsky.social:
#239047 / #239046
Would love to hear your thoughts.

Silencing during iPSC differentiation is a major but underrecognized challenge and hard to fund. We are deeply grateful to @chanzuckerberg.bsky.social for supporting this project.
Also, thanks to @brainresilience.bsky.social and @hdfcures.bsky.social for supporting my postdoctoral fellow!

Led by Marius Wernig Lab.
Thanks to our collaborators 👇
@kampmann.bsky.social @debkrov.bsky.social @florianmerkle.bsky.social @michael-e-ward.bsky.social
Ernest Arenas, Andrew Bassett, Birgitt Schuele, Leslie Thompson, & Bill Skarnes labs.
Thanks to Margaret Sutherland @chanzuckerberg.bsky.social

We shared TK4 with six independent labs.

Across protocols and cell types (neurons & microglia), the result was consistent:

CAG-WPRE (GFP) stays on.
EF1α (BFP) turns off.

Reproducible.

What about dCas9?

We tested zim3-dCas9 ± WPRE.

+WPRE: stable for 48 days.
–WPRE: silenced.

Small elements. Big consequences.

With CAG + WPRE, silencing was unaffected by insulators or integration sites.

Three novel intergenic safe harbor loci supported stable expression without disturbing endogenous genes.

We built a fully modular PiggyBac vector —
promoters, regulatory elements, insulators, and genomic loci all swappable.

During neuronal differentiation:
EF1α or loss of WPRE → silencing.

Engineering iPSCs is easy.
Keeping transgenes on is not.

Many researchers cry when expression disappears during differentiation.

That instability kills reporter gene signals, disease modeling, and CRISPR screening.

Losing sleep over #iPSC transgene silencing after differentiation?
Same here.

Bright in iPSCs.
Gone after differentiation.

Our paper in @cp-cellstemcell.bsky.social maps what actually keeps expression on👇
www.cell.com/cell-stem-ce...
🔗 Free access (50 days):
www.sciencedirect.com/science/auth...

Thrilled to see our paper out in @cp-cellstemcell.bsky.social!

Our paper on preventing transgene silencing is now online in @cp-cellstemcell.bsky.social!
🔗 Free access (50 days): www.sciencedirect.com/science/auth...

Our paper is (finally) out in Cell today!

CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis
www.cell.com/cell/fulltex...

Great collaborative effort - read more from first author @asamelson.bsky.social below:

Astrocyte induction of disease-associated microglia is suppressed by acute exposure to fAD neurons in human iPSC triple cultures Lish et al. present a co-culture system of iPSC-derived human microglia, astrocytes, and neurons to investigate intercellular communication across different environmental and genetic contexts. Transcr...

New TYP lab publication led by PhD student @alexlish.bsky.social out today in @cp-cellreports.bsky.social! We present a reproducible neuron-astrocyte-microglia tri-culture model to better reproduce intercellular interactions in Alzheimer's disease. Read more here: www.cell.com/cell-reports...

Prevention of Transgene Silencing During Human Pluripotent Stem Cell Differentiation While high and stable transgene expression can be achieved in undifferentiated pluripotent stem cells, conventional transgene expression systems are often silenced upon differentiation. Silencing occu...

Amazing team science effort, led by Marius Wernig’s group, to develop a silencing-resistant transgene cassette that maintains expression following iPSC differentiation. This was major barrier for iPSC-based experiments. Thanks to CZI for funding the research!

www.biorxiv.org/content/10.1...

Silencing of transgenes during iPSC differentiation is a frequent problem. 🧬🫢

Check out the piggyBac system below, which has resisted silencing in many protocols across many labs. 💪

Excited that our lab could contribute to this team effort led by @tuenaka.bsky.social & Marius Wernig!

Huge thanks to our amazing team:
@kampmann.bsky.social @debkrov.bsky.social @florianmerkle.bsky.social @michael-e-ward.bsky.social
Ernest Arenas, Andrew Bassett, Birgitt Schuele, Leslie Thompson, Bill Skarnes, Marius Wernig labs
Thanks to Margaret Sutherland for support via @cziscience.bsky.social

Prevention of Transgene Silencing During Human Pluripotent Stem Cell Differentiation While high and stable transgene expression can be achieved in undifferentiated pluripotent stem cells, conventional transgene expression systems are often silenced upon differentiation. Silencing occu...

Losing sleep over #iPSC transgene silencing after differentiation?
Same here — too many times😭

So we built a PiggyBac construct that resists silencing.
Expression stays ON, even after differentiation.

Our #preprint is out👇
🔗 www.biorxiv.org/content/10.1...

Supported by @cziscience.bsky.social