Reverse genetics in zebrafish by TILLING

Abstract

TILLING, for Targeting Induced Local Lesions in Genomes, is a reverse genetics strategy that identifies mutations in specific genes of interest in chemically mutagenized populations. First described in 2000 for mutation detection in Arabidopsis, TILLING is now used in a wide range of plants including soybean, rice, barley and maize as well as for animal model systems, including Arabidopsis, Drosophila, Caenorhabditis elegans, rat, medaka and zebrafish and for the discovery of naturally occurring polymorphisms in humans. This review summarizes current TILLING methodologies as they have been applied to the zebrafish, ongoing TILLING projects and resources in the zebrafish community, and the future of zebrafish TILLING.

Figure 1:

The average fertility of a cryopreserved library remains stable over several years. The library represented here was generated in the Moens lab between 2002 and 2003. Percent fertility is measured as the number of fertile eggs (developing into viable embryos) divided by the total number of eggs used in the in vitro fertilization. N refers to the number of vials of sperm thawed in each period.

Figure 2:

Similar spectra of ENU-induced mutations from forward and reverse genetic approaches in zebrafish. Mutations identified by TILLING (black bars) and in forward genetic screens (grey bars) are grouped into one of six possible classes of mutation. TILLING data are based on 982 mutations identified in the Moens lab. Forward screen data were mined from the primary literature for an arbitrary subset of zebrafish mutations that were identified through positional cloning.

Figure 3:

The number of mutations found in a fragment by TILLING with Cel1 corresponds poorly with fragment size. Thirty-six genes screened using Cel1 in the Moens lab are ranked by the size of the exon(s) that were screened (grey bars, in base pairs × 10⁻²). Black bars represent the number of mutations that were identified in those exons.