Automated live-cell single-molecule tracking in enteroid monolayers reveals transcription factor dynamics probing lineage-determining function.

Lineage transcription factors (TFs) provide one regulatory level of differentiation crucial for the generation and maintenance of healthy tissues. To probe TF function by measuring their dynamics during adult intestinal homeostasis, we established HILO-illumination-based live-cell single-molecule tracking (SMT) in mouse small intestinal enteroid monolayers recapitulating tissue differentiation hierarchies in vitro. To increase the throughput, capture cellular features, and correlate morphological characteristics with diffusion parameters, we developed an automated imaging and analysis pipeline, broadly applicable to two-dimensional culture systems.

Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors.

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue types, making them a popular choice for gene delivery in animal models and in clinical settings. In addition to therapeutic applications, rAAVs are a useful laboratory tool for delivering transgenes tailored to the researcher's experimental needs and scientific goals in cultured cells. Some examples include exogenous reporter genes, overexpression cassettes, RNA interference, and CRISPR-based tools, including those for genome-wide screens.

Towards a simple surface chemistry pre-screening method for nanoparticles to be used for drug delivery to solid tumours.

An efficient nanoparticulate drug carrier intended for chemotherapy based on intravenous administration must exhibit a long enough blood circulation time, a good penetrability into the tumour volume, as well as an efficient uptake by cancer cells. Limiting factors for the therapeutic outcome are recognition of the nanoparticles as foreign objects, which triggers nanoparticle uptake by defence organs rich in macrophages, liver and spleen, on the time-scale of accumulation and uptake in/by the tumour. However, the development of nanomedicine towards efficient nanoparticle-based delivery to solid tumours is hampered by the lack of simple, reproducible, cheap, and predictive means for early identification of promising nanoparticle formulations.

Single-molecule tracking (SMT): a window into live-cell transcription biochemistry.

How molecules interact governs how they move. Single-molecule tracking (SMT) thus provides a unique window into the dynamic interactions of biomolecules within live cells. Using transcription regulation as a case study, we describe how SMT works, what it can tell us about molecular biology, and how it has changed our perspective on the inner workings of the nucleus.