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Reverse genetic or gene-driven knockout approaches have contributed significantly to the success of model organisms for fundamental and biomedical research. Although various technologies are available for C. elegans, none of them scale very well for genome-wide application. To address this, we implemented a target-selected knockout approach that is based on random chemical mutagenesis and detection of single nucleotide mutations in genes of interest using high-throughput resequencing. A clonal library of 6144 EMS-mutagenized worms was established and screened, resulting in the identification of 1044 induced mutations in 109 Mbp, which translates into an average spacing between exonic mutations in the library of only 17 bp. We covered 25% of the open reading frames of 32 genes and identified one or more inactivating mutations (nonsense or splice site) in 84% of them. Extrapolation of our results indicates that nonsense mutations for >90% of all C. elegans genes are present in the library. To identify all of these mutations, one only needs to inspect those positions that—given the known specificity of the mutagen—can result in the introduction of a stop codon. We define these positions as nonsense introducing mutations (NIMs). The genome-wide collection of possible NIMs can be calculated for any organism with a sequenced genome and reduces the screening complexity by 200- to 2000-fold, depending on the organism and mutagen. For EMS-mutagenized C. elegans, there are only ∼500,000 NIMs. We show that a NIM genotyping approach employing high-density microarrays can, in principle, be used for the genome-wide identification of C. elegans knockouts.

OBJECTIVE: The rat is one of the most important model organisms for biomedical and pharmacological research. However, the generation of novel models for studying specific aspects of human diseases largely depends on selection for specific traits using existing rat strains, thereby solely depending on naturally occurring variation. This study aims to provide the tools to manipulate the rat genome in a more directed way. METHODS: We developed robust, automated, and scaleable reverse genetic methodology based on ENU (N-ethyl-N-nitrosourea)-driven target-selected mutagenesis. Optimal mutagenesis conditions have been determined in three different rat strains and a universal, rapid, and cost-effective dideoxy resequencing-based screening setup was established for mutation discovery. The effectiveness of the approach is illustrated by the identification of 120 induced mutations in a set of genes of interest, including six that result in unique rat knockout models due to the introduction of premature stop codons. In addition, 56 mutations were found that change amino acids, including critical residues in transmembrane domains of receptors and channels. CONCLUSIONS: The approach described here allows for the systematic generation of knockout and protein function altering alleles in the rat. The resulting induced rat models will be powerful tools for studying many aspects of a wide variety of human diseases.

The rat is one of the most extensively studied model organisms, and with its genome being sequenced, tools to manipulate gene function in vivo have become increasingly important. We here report proof of principle for target-selected mutagenesis as a reverse genetic or knockout approach for the rat.

Contains fulltext : 53181.pdf (Publisher’s version ) (Closed access) Serotonergic signaling is involved in many neurobiological processes and disturbed 5-HT homeostasis is implicated in a variety of psychiatric and addictive disorders. Here, we describe the functional characterization of the serotonin transporter (SERT) knockout rat model, that is generated by N-ethyl-N-nitrosurea (ENU)-driven target-selected mutagenesis. Biochemical characterization revealed that SERT mRNA and functional protein are completely absent in homozygous knockout (SERT-/-) rats, and that there is a gene dose-dependent reduction in the expression and function of the SERT in heterozygous knockout rats. As a result, 5-HT homeostasis was found to be severely affected in SERT-/- rats: 5-HT tissue levels and depolarization-induced 5-HT release were significantly reduced, and basal extracellular 5-HT levels in the hippocampus were ninefold increased. Interestingly, we found no compensatory changes in in vitro activity of tryptophan hydroxylase and monoamine oxidase, the primary enzymes involved in 5-HT synthesis and degradation, respectively. Similarly, no major adaptations in non-serotonergic systems were found, as determined by dopamine and noradrenaline transporter binding, monoamine tissue levels, and depolarization-induced release of dopamine, noradrenaline, glutamate and GABA. In conclusion, neurochemical changes in the SERT knockout rat are primarily limited to the serotonergic system, making this novel rat model potentially very useful for studying the behavioral and neurobiological consequences of disturbed 5-HT homeostasis.

A reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts is described and applied to create a cryopreserved resource containing knockouts for most medaka genes., We have established a reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts. A cryopreserved library of N-ethyl-N-nitrosourea (ENU) mutagenized fish was screened by high-throughput resequencing for induced point mutations. Nonsense and splice site mutations were retrieved for the Blm, Sirt1, Parkin and p53 genes and functional characterization of p53 mutants indicated a complete knockout of p53 function. The current cryopreserved resource is expected to contain knockouts for most medaka genes.

To the Editor: Although 5-HT uptake inhibitors are widely used as antidepressants, the role of 5-HT in the control of systemic blood pressure is far from clear.1 In a recent issue of Hypertension , Ni et al reported that 5-hydroxytryptamine (5-HT) transporter (SERT) expression was increased, whereas SERT function was decreased in aorta of rats with DOCA salt and Nω-nitro-L-arginine (L-NNA)-induced hypertension.2 We have observed that several 5-HT receptors are induced in leukocytes of hypertensive patients.3 These 2 observations could point at a functional response of the serotonin system …

One of the most powerful methods available to assign function to a gene is to inactivate or knockout the gene. Recently,we described the first target-selected knockout in zebrafish. Here,we report on the further improvements of this procedure,resulting in a highly efficient and easy method to do target-selected mutagenesis in zebrafish. A library of 4608 ENU-mutagenized F1 animals was generated and kept as a living stock. The DNA of these animals was screened for mutations in 16 genes by use of CEL-I-mediated heteroduplex cleavage (TILLING) and subsequent resequencing. In total,255 mutations were identified,of which 14 resulted in a premature stop codon,7 in a splice donor/acceptor site mutation,and 119 in an amino acid change. By this method,we potentially knocked out 13 different genes in a few months time. Furthermore,we show that TILLING can be used to detect the full spectrum of ENU-induced mutations in a vertebrate genome with the presence of many naturally occurring polymorphisms.

We have established a reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts. A cryopreserved library of N-ethyl-N-nitrosourea (ENU) mutagenized fish was screened by high-throughput resequencing for induced point mutations. Nonsense and splice site mutations were retrieved for the Blm, Sirt1, Parkin and p53 genes and functional characterization of p53 mutants indicated a complete knockout of p53 function. The current cryopreserved resource is expected to contain knockouts for most medaka genes.