Mike Gallagher

1/ I'm very happy to announce that the Gallagher lab will be opening at the new ASU-Banner Neurodegenerative Research Center in January 2027! We will be using iPSC-based systems to study the role of microglia (and other cells) in brain health and disease, focusing on neurodegeneration.

gallagher-lab.comGallagher Lab @ Arizona State University

4/ www.gallagher-lab.com

5/ biodesign.asu.edu/neurodegener...

biodesign.asu.eduCenter for Neurodegenerative Disease | ASU Biodesign InstituteThe ASU-Banner Neurodegenerative Disease Research Center advances the discovery of disease mechanisms for the understanding of neurodegenerative diseases.

2/ We will be employing new CRISPR-based and transgenic approaches developed in my postdoc, and are looking for interested lab techs, undergrads, MS or PhD students, postdocs, bioinformaticians, and lab managers. Please see below for the website (still a work in progress), contact me if interested,

3/ and spread the word if you know anyone who might be! The center is in the beautiful new Biodesign C building with a close-knit group of (mostly young) faculty who are highly collaborative and complementary in research focus. P.S. I have 3 years of full funding for a postdoc!

1/ Per the recent @qedscience.bsky.social discussions, I’m wondering how ppl think about principled consistency in evaluating selection-based criteria in science. E.g., any accomplishment that involves outcompeting other ppl (grad school admissions, publishing, grant apps, awards/fellowships, etc)

5/ and randomness involved in many such “rankings”. This is addressed to those who abhor and reject ALL metrics/rankings, not those who only question specific ranking methodologies. Thoughts? @odedrechavi.bsky.social @richardsever.bsky.social @mbeisen.bsky.social @arjunraj.bsky.social

6/ @epididymosome.bsky.social @philipcball.bsky.social @michaelzlin.bsky.social

3/ such as QED 1%?. If the issue is that the criteria for winning are questionable, that’s fine, but some ppl seem to think that ANY ranking or metric is intrinsically antithetical to science, and therefore should be eliminated. I’m just thinking out loud - everything in life involves competition,

4/ and we don’t roll our eyes at the student who got into grad school, or the postdoc who got a TT position, just because it’s another “metric” or “ranking”, even though in reality it is! We congratulate them bc we’re happy that they achieved a goal for themselves, despite the obvious subjectivity

2/ it is common to congratulate ppl on these accomplishments, even though it implicitly acknowledges that they outcompeted X% of other candidates. If it is OK to congratulate someone who wins a fellowship as a direct result of scoring in the X%, why is it not OK to do this in other competitions,

1/ Happy to share important work done with my co-author Andrew Khalil in the labs of Rudolf Jaenisch @whiteheadinstitute.bsky.social @mit.edu and David Mooney @harvard.edu @wyssinstitute.bsky.social trying to assess and fix the major problem of transgene silencing in human ESC/iPSC based work

4/ We compared multiple existing systems, tried to fix the ones that don't work, and developed a new system that overcomes many existing problems, using #microglia and #macrophages as test cases #Immunotherapy #neuroinflammation LINK:https://www.biorxiv.org/content/10.1101/2025.10.17.683097v1

biorxiv.orgSystematic characterization of existing and novel inducible transgenic systems in human pluripotent stem cells after prolonged differentiationThe ability to control transgene expression both temporally and quantitatively in human-relevant cells and tissues is a cornerstone of biomedical research. Additionally, precise transgene control is c...

5 Nov 2025

Oops, here’s the right link www.biorxiv.org/content/10.1...

19/ strangely, leaky SALL1 is not due to unwanted splicing in the absence of drug, but rather the unspliced pre-mRNA somehow escapes the nucleus and is translated in-frame to make the full-length protein. We still don't know how this works.

20/ in summary, this "iPSC-Xon" system overcomes critical limitations in current transgene systems in hPSC-based work, and will benefit from additional improvements. I'm on the job market and would love to bring and improve this technology to other researchers!

17/ we also show that we can get inducible gene editing with Xon hPSCs, but only if sgRNAs are introduced transiently (panels C-E). Cas9 must have enough leakiness for maximal editing with constitutive lentiviral sgRNA introduced into hPSCs (A-B)

18/ we thought larger transgenes (Cas9 is ~4kb) might be more prone to disrupting the Xon splicing switch, so we tried the ~4kb SALL1 TF, a critical driver of microglia homeostatic function. In contrast with EGFP, Xon-SALL1 is fully leaky and shows aberrant splicing patterns.

15/ a couple important points here: in addition to resisting silencing and being highly tunable, Xon adds only 24aa N-terminal tag (see below where we reduce it to 1aa) and needs only low nM drug doses, whereas degrons add ~500bp tags to BOTH termini to be effective, and need low uM drug doses.

16/ what about other genes? Here we show Xon works for inducible control of GRN expression, and we can overexpress GRN in iPSC-derived microglia, which already express boatloads of it. P2A peptides improve both GRN and SMAD, and importantly reduce N-terminal tag to a single aa.

13/ Here's the commonly used H1 ESC background, differentiated into microglia with an embryoid body protocol (we used EB and monolayer)

14/ a note of caution is that while background EGFP is minor compared to even the lowest drug induced expression levels, this may need mitigation when using TFs or other strong cell state regulators

11/ again with AAVS1-targeted EGFP, what we saw was remarkable: first time we've ever seen robust inducibility after long differentiations, this time 4-7 weeks, across hPSC backgrounds and differentiation protocols!

12/ quantification shows how tunable this system really is! Below the 100nM dose, linear correlations between drug dose and EGFP is basically perfect both before and after differentiation

9/ we also looked at leakiness, and leaky EGFP varied by system, with Tet-On being pretty tightly controlled (although silenced later on), and degrons showing variability. Note lack of degron tunability in panel C.

pmc.ncbi.nlm.nih.govRegulated control of gene therapies by drug-induced splicingSo far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The ...

10/ we then adapted the recently described "Xon" splicing switch, from this paper: pmc.ncbi.nlm.nih.gov/articles/PMC... Could this approach work in hPSCs, resist silencing and allow for robust inducibility in differentiated microglia and macrophages?

8/ We next tested degrons, as this have been used successfully for transgene expression specifically in fully differentiated cells. However, their leakiness, tunability, and inducibility hasn't been thoroughly tested.

8b/ as you can see above, degrons vary in performance, but all show no tunability and only modest inducibility after differentiation.

6/ we then tried to fix this by adding a Tet-Off-TET1 fusion protein, as DNA methylation has been proposed to underlie Tet-On silencing. Here's the idea, with existing systems (top) getting silenced before DOX is added, and new systems (bottom) we hoped would fix it

7/ all approaches used EGFP after AAVS1 safe harbor targeting. While these systems worked in hPSCs (albeit with slower kinetics), they still got silenced during differentiation

5/ First we showed that Tet-On-based transgenes are silenced early during microglia or macrophage differentiation, regardless of transgene delivery method, transgene components, hPSC background, or differentiation protocol

2/ we cannot currently alter genes and pathways in many differentiated hPSC-derived cell types without potential effects on the differentiation itself, due to transgene silencing, lack of transgene tunability, and toxicity or unwanted effects of electroporation and transduction #ESC #iPSC #synbio

3/ this is a major problem for both basic biomedical research in disease-relevant cells/tissues, as well as CRISPR screens and other systematic gene/pathway dissections that must be done with careful control at the right stage of differentiation. #bioengineering #genomics #stemcells