Edwin Cuppen, Hubrecht Lab, Utrecht, The Netherlands | external teacher


Target-selected mutagenesis: Making knockouts in zebrafish
As of today, no pluripotent stem cells are available for zebrafish, excluding the possibility to generate knockouts analagous as for mice using homologous recombination in ES cells. However, we exploited the efficient chemical ENU (N-ethyl-N-nitrosourea)-based mutagenesis and the increasing efficiency of (point-) mutation discovery to show a proof of principle for generating zebrafish knockouts by target-selected mutagenesis (also known as Targeting Induced Local Lesions IN Genomes or TILLING) and we reported the first zebrafish knockout in 2002 (Wienholds et al. (2002), Science 297: 99-102).

Briefly, we mutagenize male founder fish using ENU, breed these animals with wild-type females, resulting in a large population of F1 fish that carry many independent heterozygous point mutations. DNA is isolated from these fish and used for high-throughput mutation discovery in the genomic regions harboring genes of interest (see Figure 1).

Figure 1: Schematic outline of the target-selected mutagenesis procedure.

In this course, we will discuss several aspects of the technology, including ENU-mutagenesis, living and cryopreserved F1 libraries, and different technologies for mutation discovery. Furthermore, we will address issues for both small- and large-scale implementation in your own lab and bioinformatic tools that we have developed (e.g. http://limstill.niob.knaw.nl) for facilitating various steps of the procedure.

More information and protocols can be found on: http://www.niob.knaw.nl/researchpages/cuppen

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