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Embryonic stem cells (left) and with a chemically induced knockout of a gene essential for cell growth and survival (right).






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FMI

September 1, 2015

An important shortcut to models of disease

Marc Bühler and his group at the FMI have developed a new and more efficient way to generate embryonic stem cells in which a given gene can be deleted at will, so called conditional knockout cells. They used the engineered nuclease TALEN and a reporter to enrich cells that have integrated defined sequences into the genome through homologous recombination. This refined method makes the use of animals for the generation of conditional knockout cells obsolete. It will also reduce the number of animals required to establish mouse models of disease dramatically. Their new method is therefore in line with 3R efforts around the world.

Mouse strains lacking certain proteins, so called knockout mice, are valuable research models to understand the mechanisms of disease. Unfortunately, the breeding of these mouse strains is laborious, intricate and time consuming. This is particularly true for conditional knockout mice that allow you to study the function of a protein in a clearly defined cell type, e.g. in the epithelial cells of the gut, at a specific time point during development, e.g. in the adult.

The recent advent of new genome editing tools such as CRISPR/Cas9 and TALENs has allowed to disrupt genes in animal cells kept in culture (see box). However, it has not yet contributed to simplifying the generation of conditional knockouts. These conditional knockouts depend on the simultaneous integration of two DNA fragments at the same time (something that happens very rarely), and the expression of a protein that is required to delete the respective gene upon a chemical stimulus.

Matyas Flemr, a postdoctoral fellow in Marc Bühler’s group at the Friedrich Miescher Institute for Biomedical Research has now found a way to establish conditional mouse embryonic stem cells in one single step in vitro. “We introduced a reporter gene into the setup that would allow us to screen for cells with successful genomic integrations,” explained Flemr, “assuming that once the TALEN system and the homologous recombination-mediated DNA repair pathway were activated, the conditional knockout fragments but not the reporter gene would be integrated. It was kind of an adventurous assumption.”

But this strategy turned out to work very well. TALENs stimulate the knockin of DNA fragments by homologous recombination overall, and the reporter allowed the enrichment of the cells where homologous recombination had happened. “I was most surprised that we obtained homozygous conditional knockout cells in one go”, comments Bühler. “So far, this required live animals, complicated embryo manipulation techniques, and was very time consuming. The conventional protocols take at least one year and involve multiple crossings. With the protocols and reagents that we have established, we have results in less than 3 weeks and with no animals involved. Furthermore, our approach is amenable to repeated knockins in the same cell line and therefore it allows us to knockout several genes in a time frame of weeks, compared to several months by conventional techniques.”

Using this system, the scientists are now able to establish genetically modified mouse embryonic stem cells in vitro, replacing the use of animals entirely. Mouse embryonic stem cells are pluripotent and they can thus be differentiated into different cell types. Therefore, animals could in principle no longer be required to obtain cellular models of diseases. “I expect that the simple system that we introduced to the community will also reduce the time and the resources needed to generate conditional knockout mice dramatically,” commented Bühler. “We no longer need to cross and breed animals extensively to obtain an interesting disease model to perform in vivo studies. This is a beautiful example of how we can develop new, and refine existing methods to reduce the use of laboratory animals.”

CRISPR/Cas9 and TALENs
CRISPR/Cas9 and TALENs are engineered bacterial nucleases that induce double strand breaks at defined positions in the genome. These breaks are then repaired by the cellular repair machineries, in some cases based on a template present in the cell. If the template bears mutations or deletions, the repair machinery will introduce these changes into the genome. Scientists have now used these systems to purposefully inactivate genes or introduce mutations in a variety of species. In the CRISPR/cas9 system, the nuclease is guided to the right position by a piece of RNA, which is complementary to the gene of interest. TALENs recognize the DNA sequence through their amino acid sequence. The double-strand breaks are then repaired using a template DNA.

3Rs in animal research
The 3Rs stand for:
  • Reducing the number of animals used in studies;
  • Refining study methods;
  • Replacing animal studies with new methods
They not only encourage alternatives to animal testing but also aim to improve animal welfare and scientific quality. The 3R principles are an integral part of the Swiss animal welfare law.

Original publication
Flemr M, Bühler M (2015) Single-step generation of conditional knockout mouse embryonic stem cells. Cell Rep. 12:709-16

On Marc Bühler
Marc Bühler is a Senior group leader at the FMI. His team is investigating epigenetic processes in yeast and animals. By focusing on the interplay between RNAs and chromatin, his work combines two cutting-edge research areas.
» More about Marc Bühler

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