A large-scale zebrafish gene knockout resource for the genome-wide study of gene function

Abstract

With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1's predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ∼0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome.

An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

“…Large-scale mapped mutant libraries have transformed research in bacteria (Baba et al, 2006;Goodman et al, 2009), yeast (Winzeler et al, 1999;Giaever et al, 2002), animals (Venken et al, 2011;Varshney et al, 2013), and land plants (Alonso et al, 2003;May et al, 2003;McCarty et al, 2005;Zhang et al, 2006;Hsing et al, 2007;Bragg et al, 2012;Belcher et al, 2015). An analogous stably maintained library of Chlamydomonas insertional mutants with known insertion sites would have a similarly transformative impact on the ease of elucidating gene functions in this organism.…”

Shroom3 contributes to the maintenance of the glomerular filtration barrier integrity

“…Furthermore, these loci collectively explain only a small percentage of disease heritability, indicating the need to identify alternative sources of ''missing'' heritability (Manolio et al 2009;Chatterjee et al 2013). Genomic infrastructure available for animal models provides a powerful complementary tool to GWAS (Aitman et al 2008;Geurts et al 2009;Cox and Church 2011;Lewis and Tomlinson 2012;Atanur et al 2013;Varshney et al 2013). For example, mapping quantitative trait loci (QTL) in mammals and identifying natural mutations in syntenic regions of animal disease models have provided functional support for human risk loci in multiple diseases (Aitman et al 2008).…”

Les modèles animaux de l’anémie de Fanconi

“…Enfin, le poisson zèbre permet la réalisation de diverses études de mutagenèse pour identifier et cloner de nouveaux gènes Fanc non identifiés encore (Figure 2). Il pourrait également aider à concevoir et caractériser un large panel de nouvelles molécules thérapeutiques pour l'anémie de Fanconi [5,57]. Pour conclure, la recherche sur l'anémie de Fanconi a grandement progressé dans sa connaissance de la voie FA-BRCA grâce à l'étude de la conservation des protéines FANC et la compréhension des différences de phénotypes entre l'homme et d'autres systèmes in vivo.…”