Biology subjects

Gallagher, M. D.

Publications and source records attributed to Gallagher, M. D..

3 recordsLinked to original sources

bioRxiv2025

Systematic characterization of existing and novel inducible transgenic systems in human pluripotent stem cells after prolonged differentiation

The 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 crucial for optimizing human cell-based gene therapies. Human pluripotent stem cells (hPSCs) have facilitated major advances in disease modeling and the potential for regenerative medicine. Still, they are significantly limited by the lack of inducible transgenic systems that avoid silencing but maintain robust inducibility after differentiation to defined cell lineages. Here we systematically characterize the leakiness, inducibility, and tunability of multiple existing and novel transgenic systems in hPSCs and differentiated macrophages and microglia. Notably, we report the application of a small molecule-mediated splicing switch (Xon) that allows for tunable transgene expression both before and after differentiation, without the large protein tags required for current state-of-the-art degron-based methods. We use Xon to achieve tight control of reporter genes, overexpression of multiple neurodegeneration-associated genes, and Cas9-mediated genome editing. We also characterize the current limitations of this system and describe approaches that can alleviate some of these limitations. By assessing multiple transgenic systems whose inducibility spans the transcriptional, post-transcriptional, and post-translational levels, we highlight and improve upon a major technical challenge that hinders basic, translational, and clinical research in physiological human-based systems.

bioengineering↗

bioRxiv2024

Optical genome mapping enables accurate repeat expansion testing

Short tandem repeats (STRs) are amongst the most abundant class of variations in human genomes and are meiotically and mitotically unstable which leads to expansions and contractions. STR expansions are frequently associated with genetic disorders, with the size of expansions often correlating with the severity and age of onset. Therefore, being able to accurately detect the total repeat expansion length and to identify potential somatic repeat instability is important. Current standard of care (SOC) diagnostic assays include laborious repeat-primed PCR-based tests as well as Southern blotting, which are unable to precisely determine long repeat expansions and/or require a separate set-up for each locus. Sequencing-based assays have proven their potential for the genome-wide detection of repeat expansions but have not yet replaced these diagnostic assays due to their inaccuracy to detect long repeat expansions (short-read sequencing) and their costs (long-read sequencing). Here, we tested whether optical genome mapping (OGM) can efficiently and accurately identify the STR length and assess the stability of known repeat expansions. We performed OGM for 85 samples with known clinically relevant repeat expansions in DMPK, CNBP and RFC1, causing myotonic dystrophy type 1 and 2 and cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), respectively. After performing OGM, we applied three different repeat expansion detection workflows, i.e. manual de novo assembly, local guided assembly (local-GA) and molecule distance script of which the latter two were developed as part of this study. The first two workflows estimated the repeat size for each of the two alleles, while the third workflow was used to detect potential somatic instability. The estimated repeat sizes were compared to the repeat sizes reported after the SOC and concordance between the results was determined. All except one known repeat expansions above the pathogenic repeat size threshold were detected by OGM, and allelic differences were distinguishable, either between wildtype and expanded alleles, or two expanded alleles for recessive cases. An apparent strength of OGM over current SOC methods was the more accurate length measurement, especially for very long repeat expansion alleles, with no upper size limit. In addition, OGM enabled the detection of somatic repeat instability, which was detected in 9/30 DMPK, 23/25 CNBP and 4/30 RFC1 samples, leveraging the analysis of intact, native DNA molecules. In conclusion, for tandem repeat expansions larger than [~]300 bp, OGM provides an efficient method to identify exact repeat lengths and somatic repeat instability with high confidence across multiple loci simultaneously, enabling the potential to provide a significantly improved and generic genome-wide assay for repeat expansion disorders.

genomics↗

bioRxiv2020

The structural variation landscape in 492 Atlantic salmon genomes

Structural variants (SVs) are a major source of genetic and phenotypic variation, but remain challenging to accurately type and are hence poorly characterized in most species. We present an approach for reliable SV discovery in non-model species using whole genome sequencing and report 15,483 high-confidence SVs in 492 Atlantic salmon (Salmo salar L.) sampled from a broad phylogeographic distribution. These SVs recover population genetic structure with high resolution, include an active DNA transposon, widely affect functional features, and overlap more duplicated genes retained from an ancestral salmonid autotetraploidization event than expected. Changes in SV allele frequency between wild and farmed fish indicate polygenic selection on behavioural traits during domestication, targeting brain-expressed synaptic networks linked to neurological disorders in humans. This study offers novel insights into the role of SVs in genome evolution and the genetic architecture of domestication traits, along with resources supporting reliable SV discovery in non-model species.

genomics↗