Practical Course HP-F5: Advanced Methods in Cell Biology

Type: Practical Course with Student Seminars

Date: 13.-31. January 2020

Locations: TP3 Division Labs

Hosts/Supervisors: Frank Lyko, Vitor Coutinho Carneiro; Michael Boutros, Florian Heigwer, Fillip Port (organizer contact: u.hardeland@dkfz.de)

Topics:

  • A)  Detection and experimental modulation of DNA methylation in human cancer cells
  • B)  RNA interference as a tool to study gene function
  • C) Tissue-specific CRISPR/Cas9 knock-out screening in Drosophila

Content:

general: Cell culture, immunofluorescence microscopy, Western blotting, cDNA transfection, bisulfite sequencing, RNAi screening, high-throughput data analysis

details for topic A): DNA methylation at gene promoters and other regulatory elements constitutes an important and probably the best-studied epigenetic modification. In this part of the course, we will investigate DNA methylation changes upon cell differentiation by bisulfite sequencing and the implications of these changes on transcription (RT-PCR) and expression (semiquant. Western blotting).

details for topic B): RNA interference (RNAi) has become an important method for the characterization of gene function. RNAi allows the silencing of specific genes through introduction of short double-stranded RNAs homologous to endogenous mRNAs. As part of the course, participants will learn how to perform RNAi experiments in Drosophila cells and analyze phenotypic changes using image based single cell profiling. We will perform a small scale RNAi screen to analyze morphological phenotypes after perturbation of a number of signaling pathways, which are known to be involved in the development of various cancers. The course will include hands-on practice in data analysis of high-content profiling experiments using state of the art computational methods.

details for topic C): CRISPR/Cas9 genome engineering permits targeted genome manipulations with unprecedented ease and precision. We will use a novel system that permits activation of the CRISPR system only in certain tissues within a multicellular
organism. Using Drosophila melanogaster as a model, participants will knock-out candidate genes in a subset of epithelial cells and analyse the resulting phenotypes. In particular, we will use novel knock-in reporter lines as well as immunohistochemistry to analyse possible effects of gene disruption on the endogenous Wnt pathway.

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