Entschlüsselung der molekularen Netzwerke, die das neurogene Potenzial von Astrozyten im erwachsenen Mausgehirn bestimmen
Entwicklungsneurobiologie
Zusammenfassung der Projektergebnisse
During the first part of the project, I successfully employed the previously suggested strategy for CRISPR-based generation of a genetic barcode in mouse cells in vitro and the mouse brain in vivo coupled to an RNA-seq readout. However, clone and lineage reconstruction using computational methods proved difficult even in a control in vitro experiment and in vivo barcoding in the mouse brain resulted in less than 6% of barcoded cells. While the idea of applying CRISPR for barcoding and lineage reconstruction remains attractive, its practical application for the mouse brains remains challenging and requires dedicated fine tuning to that model system. In particular, various biological and technical parameters including variable cell division rates, skewed mutational outcomes, target site dropouts and different sequencing strategies, have a large impact on the accuracy of the reconstructed lineages. With that in mind and due to time as well as technical constraints, we decided to employ a more practical barcoding strategy and apply it to the developing mouse brain as a first proof-of-principle study. The second part of the project focused on the development of BrainTrace, an approach that involves genetic barcoding of radial progenitor cells in vivo coupled to lineage tracing using single cell transcriptomics. I constructed a highly diverse lentiviral barcode library that was delivered into the developing mouse brain and barcodes together will transcriptomes could be read out using single cell RNA-seq. I successfully demonstrated the first proof-of-principle application of this methodology in the mouse brain and we are currently writing a manuscript summarizing our findings about regional and fate distributions of clonally related cells. Future developments will focus on Cre-inducible and cell type specific barcoding in the developing and adult mouse brain, in situ RNA-seq readout using Spatial Transcriptomics as well as CRISPR-mediated perturbations coupled to barcoding and clonal tracing.
Projektbezogene Publikationen (Auswahl)
- (2017): STRT-seq-2i: dual-index 5ʹ single cell and nucleus RNA-seq on an addressable microwell array. Scientific Reports 7: 6327
Hochgerner, H, Lönnerberg, P, Hodge, R, Mikes, J, Heskol, A, Hubschle, H, Lin, P, Picelli, S, La Manno, G, Ratz, M, Dunne, J, Husain, S, Lein, E, Srinivasan, M, Zeisel, A, Linnarsson, S
(Siehe online unter https://doi.org/10.1038/s41598-017-16546-4) - (2017): Two-Color 810 nm STED Nanoscopy of Living Cells with Endogenous SNAP-Tagged Fusion Proteins. ACS Chemical Biology 13: 475-480
Butkevich, AN, Ta, H, Ratz, M, Stoldt, S, Jakobs, S, Belov, VN, Hell, SW
(Siehe online unter https://doi.org/10.1021/acschembio.7b00616) - (2018): Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems. Nature Communications 9: 3281
Masullo, LA, Boden, A, Pennacchietti, F, Coceano, G, Ratz, M, Testa, I
(Siehe online unter https://doi.org/10.1038/s41467-018-05799-w)
