Functional genomics in mammalian cells: applications to cancer- and stem cell- biology
Previous and Current Research
Sequencing of whole genomes has provided new perspectives into the blueprints of diverse organisms. Knowing the sequences, however, does not always tell us much about the function of the genes that regulate development and homeostasis. Our laboratory is using different strategies to dissect gene function in mammalian cells relevant to cancer biology and stem cell research.
Loss-of-function screens in mammalian cells with esiRNA
RNA interference is becoming the method of choice for gene function analysis in cells and whole organisms. Our lab has developed innovative technology to perform large scale RNAi experiments in mammalian cells and in mice. We use enodibonuclease prepared siRNA (esiRNA) for the efficient and specific knock down in genome wide screens to identify and characterize genes relevant to cancer biology and differentiation.
Normal and malignant haematopoiesis
Haematopoietic and endothelial cells arise from a common precursor, commonly referred to as the haemangioblast. The decision whether the cells differentiate into the blood- or vessel- lineage seems to correlate with the expression of certain "master switch genes". One of these important genes for lineage specification is the runx1 gene. Runx1 seems to participate in the fate decision in favor of haematopoietic development. Its pivotal role during development makes runx1 also a good candidate to play an important role in adult stem cells. Interestingly, runx1 is also one of the most frequently mutated genes in human leukemias, supporting that runx1 plays an important role in the adult. We study the role of runx1 during development and in adult mice. A better understanding of this master switch regulator may help to understand, how cells that have a common origin take on a specific fate in the body to build an organ and may also help to understand the development of certain leukemias.
Advanced genome engineering
The need for fluent and precise genomic manipulation strategies has been exacerbated by the increasing pace of published genome sequences. Beyond the reading of complete genomes, the precise manipulation of the encoded information is becoming more important in modern molecular biology. Site specific recombinases are prominent genetic engineering tools that allow the genetic manipulation of whole organisms. In order to expand the usefulness of these enzymes we are designing novel recombinases through directed molecular evolution and test them for their usefulness in biology and medicine.
Future Projects and Goals
- Extend the esiRNA technology to long non-coding RNAs,
- Perform RNAi screens in mouse ES cells to identify and characterize factors involved in self renewal and differentiation,
- Perform phenotypic profiling in primary human cells to identify and characterize candidate genes with therapeutic potential,
- Develop and apply tools that allow flexible and precise genomic manipulations
Methodological and Technical Expertise
- Large scale RNAi screens
- Live cell microscopy
- directed molecular evolution
- site-specific recombination
- Ion Torrent sequencing
Karpinski J, Hauber I, Chemnitz J, Schäfer C, Paszkowski-Rogacz M, Chakraborty D, Beschorner N, Hofmann-Sieber H, Lange UC, Grundhoff A, Hackmann K, Schrock E, Abi-Ghanem J, Pisabarro MT, Surendranath V, Schambach A, Lindner C, van Lunzen J, Hauber J, Buchholz F.
Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity.
Nat Biotechnol. 34(4):401–9. (2016)
Krastev DB, Slabicki M, Paszkowski-Rogacz M, Hubner NC, Junqueira M, Shevchenko A, Mann M, Neugebauer KM, Buchholz F
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A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity.
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HIV-1 proviral DNA excision using an evolved recombinase.Science 316: 1912-1915. (2007)
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An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division.
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