I believe the top down approach is more efficient and economical. It is also notable that the final characterization of the behavioral alteration should include multiple tests that tap into the same function but using different methods.

For example, testing relational learning in rodents can be achieved using the Morris water maze spatial learning task as well as the context GDC-0199 purchase dependent fear conditioning task [12]. While such well developed tasks do not yet exist for the zebrafish, the principles are the same: tasks with different performance demands tapping into the same principle brain function allow the experimenter to exclude performance alterations and focus on the main goal: in this example relational learning mechanisms. The last topic I will briefly consider is what forward genetic method to chose. The most frequently employed method has been ethyl nitroso-urea (ENU) mutagenesis [29]. While this method is highly efficient in inducing single point mutations with a relatively homogeneous and full coverage of the entire genome, its disadvantage has been the labor intensive linkage analysis based positional cloning method that is required for the identification

of the gene that carries the mutation. Linkage analysis requires multiple generations of breeding a large number of fish and positional cloning is also a labor intensive molecular biology technique. While ENU is still the most prevalent approach in the zebrafish forward genetics literature, ON-01910 clinical trial alternative mutagenesis methods

are also becoming a reality. The main advantage of these newer methods is that they allow rapid identification of the mutated gene and/or allow the precise targeting of the mutation to known sequences. Viral-vector mediated, or insertional, mutagenesis was introduced several years ago [30]. Because the mutation is induced by insertion of a non-native nucleotide sequence into the zebrafish genome and because this sequence is known, identification of the gene with the inserted sequence can be achieved in a single step. Meloxicam Another promising approach that has recently been introduced is the TALEN (transcription activator-like effector nuclease) system. TALENs are artificial restriction endonuclease-like enzymes. These enzymes are generated by fusing a transcription activator-like effector (TALE) DNA binding domain to a DNA cleavage domain. The method has been optimized for the zebrafish [31••] and has been claimed to allow one to target practically any desired zebrafish gene in an efficient manner. This essentially reverse genetic method may be utilized for forward genetics too because the zebrafish genome has been sequenced and suspected, that is, previously not cloned genes and uncharacterized genes, can now be targeted en masse and phenotypically characterized.