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2 results on '"Marie-Christin Leitner"'

1. An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

Author

Pooran Singh Dewari, Benjamin Southgate, Katrina Mccarten, German Monogarov, Eoghan O'Duibhir, Niall Quinn, Ashley Tyrer, Marie-Christin Leitner, Colin Plumb, Maria Kalantzaki, Carla Blin, Rebecca Finch, Raul Bardini Bressan, Gillian Morrison, Ashley M Jacobi, Mark A Behlke, Alex von Kriegsheim, Simon Tomlinson, Jeroen Krijgsveld, and Steven M Pollard

Subjects
neural stem cell, CRISPR, epitope tagging, stem cells, genome editing, transcription factors, Medicine, Science, Biology (General), QH301-705.5
Abstract

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5–30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.

Published
2018
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2. De novo PITX1 expression controls bi-stable transcriptional circuits to govern self-renewal and differentiation in squamous cell carcinoma

Author

Martyna Okuniewska, Ana Sastre-Perona, Marie-Christin Leitner, Shane Meehan, Markus Schober, and Steven Hoang-Phou

Subjects
Transcription, Genetic, Mice, Nude, Cell fate determination, Biology, Article, Transcriptome, 03 medical and health sciences, Basal (phylogenetics), Kruppel-Like Factor 4, Mice, 0302 clinical medicine, SOX2, stomatognathic system, Cancer stem cell, Genetics, Tumor Cells, Cultured, Animals, Humans, Paired Box Transcription Factors, Basal cell, Transcription factor, 030304 developmental biology, Cell Proliferation, 0303 health sciences, fungi, Cell Differentiation, Cell Biology, Cell biology, Gene Expression Regulation, Neoplastic, KLF4, embryonic structures, Carcinoma, Squamous Cell, Molecular Medicine, Female, sense organs, 030217 neurology & neurosurgery
Abstract

Summary Basal tumor propagating cells (TPCs) control squamous cell carcinoma (SCC) growth by self-renewing and differentiating into supra-basal SCC cells, which lack proliferative potential. While transcription factors such as SOX2 and KLF4 can drive these behaviors, their molecular roles and regulatory interactions with each other have remained elusive. Here, we show that PITX1 is specifically expressed in TPCs, where it co-localizes with SOX2 and TRP63 and determines cell fate in mouse and human SCC. Combining gene targeting with chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomic analyses reveals that PITX1 cooperates with SOX2 and TRP63 to sustain an SCC-specific transcriptional feed-forward circuit that maintains TPC-renewal, while inhibiting KLF4 expression and preventing KLF4-dependent differentiation. Conversely, KLF4 represses PITX1, SOX2, and TRP63 expression to prevent TPC expansion. This bi-stable, multi-input network reveals a molecular framework that explains self-renewal, aberrant differentiation, and SCC growth in mice and humans, providing clues for developing differentiation-inducing therapeutic strategies.

Published
2019

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