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1.  Conditional Gene-Trap Mutagenesis in Zebrafish 
Zebrafish has become a widely used model for analysis of gene function. Several methods have been used to create mutations in this organism and thousands of mutant lines are available. However, all the conventional zebrafish mutations affect the gene in all cells at all time, making it difficult to determine tissue-specific functions. We have adopted a FlEx Trap approach to generate conditional mutations in zebrafish by gene trap mutagenesis. Combined with appropriate Cre or Flp lines, the insertional mutants not only allow spatial and temporal specific gene inactivation, but also permit spatial and temporal specific rescue of the disrupted gene. We provide experimental details on how to generate and use such mutations.
PMCID: PMC4129463  PMID: 24233792
zebrafish; mutagenesis; gene-trap; Cre; Flp
2.  Targeted Overexpression of CKI-Insensitive Cyclin-Dependent Kinase 4 Increases Functional β-Cell Number Through Enhanced Self-Replication in Zebrafish 
Zebrafish  2013;10(2):170-176.
β-Cells of the islet of Langerhans produce insulin to maintain glucose homeostasis. Self-replication of β-cells is the predominant mode of postnatal β-cell production in mammals, with about 20% of rodent β cells dividing in a 24-hour period. However, replicating β-cells are rare in adults. Induction of self-replication of existing β-cells is a potential treatment for diabetes. In zebrafish larvae, β-cells rarely self-replicate, even under conditions that favor β-cell genesis such overnutrition and β-cell ablation. It is not clear why larval β-cells are refractory to replication. In this study, we tested the hypothesis that insufficient activity of cyclin-dependent kinase 4 may be responsible for the low replication rate by ectopically expressing in β-cells a mutant CDK4 (CDK4R24C) that is insensitive to inhibition by cyclin-dependent kinase inhibitors. Our data show that expression of CDK4R24C in β-cells enhanced β-cell replication. CDK4R24C also dampened compensatory β-cell neogenesis in larvae and improved glucose tolerance in adult zebrafish. Our data indicate that CDK4 inhibition contributes to the limited β-cell replication in larval zebrafish. To our knowledge, this is the first example of genetically induced β-cell replication in zebrafish.
PMCID: PMC3673610  PMID: 23544990
3.  A gain-of-function screen in zebrafish identifies a guanylate cyclase with a role in neuronal degeneration 
Molecular genetics and genomics : MGG  2009;281(5):10.1007/s00438-009-0428-8.
Manipulation of gene expression is one of the most informative ways to study gene function. Genetic screens have been an informative method to identify genes involved in developmental processes. In the zebrafish, loss-of-function screens have been the primary approach for these studies. We sought to complement loss-of-function screens using an unbiased approach to overexpress genes with a Gal4-UAS based system, similar to the gain-of-function screens in Drosophila. Using MMLV as a mutagenic vector, a cassette containing a UAS promoter was readily inserted in the genome, often at the 5′ end of genes, allowing Gal4-dependent overexpression. We confirmed that genes downstream of the viral insertions were overexpressed in a Gal4-VP16 dependent manner. We further demonstrate that misexpression of one such downstream gene gucy2F, a membrane-bound guanylate cyclase, throughout the nervous system results in multiple defects including a loss of forebrain neurons. This suggests proper control of cGMP production is important in neuronal survival. From this study we propose that this gain-of-function approach can be applied to large-scale genetic screens in a vertebrate model organism and may reveal previously unknown gene function.
PMCID: PMC3814131  PMID: 19221799
insertional mutagenesis; forward genetic screen; Gal4-VP16; guanylate cyclase
4.  Nutrient Excess Stimulates β-Cell Neogenesis in Zebrafish 
Diabetes  2012;61(10):2517-2524.
Persistent nutrient excess results in a compensatory increase in the β-cell number in mammals. It is unknown whether this response occurs in nonmammalian vertebrates, including zebrafish, a model for genetics and chemical genetics. We investigated the response of zebrafish β-cells to nutrient excess and the underlying mechanisms by culturing transgenic zebrafish larvae in solutions of different nutrient composition. The number of β-cells rapidly increases after persistent, but not intermittent, exposure to glucose or a lipid-rich diet. The response to glucose, but not the lipid-rich diet, required mammalian target of rapamycin activity. In contrast, inhibition of insulin/IGF-1 signaling in β-cells blocked the response to the lipid-rich diet, but not to glucose. Lineage tracing and marker expression analyses indicated that the new β-cells were not from self-replication but arose through differentiation of postmitotic precursor cells. On the basis of transgenic markers, we identified two groups of newly formed β-cells: one with nkx2.2 promoter activity and the other with mnx1 promoter activity. Thus, nutrient excess in zebrafish induces a rapid increase in β-cells though differentiation of two subpopulations of postmitotic precursor cells. This occurs through different mechanisms depending on the nutrient type and likely involves paracrine signaling between the differentiated β-cells and the precursor cells.
PMCID: PMC3447891  PMID: 22721970
5.  Generating Conditional Mutations in Zebrafish Using Gene-trap Mutagenesis 
Methods in cell biology  2011;104:1-22.
While several mutagenesis methods have been successfully applied in zebrafish, these mutations do not allowed tissue- or temporal-specific functional analysis. We have developed a strategy that will allow tissue- or temporal-specific disruption of genes in zebrafish. This strategy combines gene trap mutagenesis and FlEx modules containing target sites for site specific recombinases. The gene trap cassette is highly mutagenic in one orientation and non-mutagenic in the opposite orientation, with different fluorescent proteins as indicators of the orientation. The inclusion of the FlEx modules allows two rounds of stable inversion mediated by the Cre and Flp recombinases. This gene trap cassette can be easily delivered via transposons. Through large-scale community-wide efforts, broad genome coverage can be obtained. This should allow investigation of cell/tissue specific gene function of a wide-range of genes.
PMCID: PMC3438898  PMID: 21924154
6.  PhiC31 integrase induces efficient site-specific excision in zebrafish 
Transgenic research  2010;20(1):183-189.
Site-specific recombinases catalyze recombination between specific targeting sites to delete, insert, invert, or exchange DNA with high fidelity. In addition to the widely used Cre and Flp recombinases, the phiC31 integrase system from Streptomyces phage may also be used for these genetic manipulations in eukaryotic cells. Unlike Cre and Flp, phiC31 recognizes two heterotypic sites, attB and attP, for recombination. While the phiC31 system has been recently applied in mouse and human cell lines and in Drosophila, it has not been demonstrated whether it can also catalyze efficient DNA recombination in zebrafish. Here we show that phiC31 integrase efficiently induces site-specific deletion of episomal targets as well as chromosomal targets in zebrafish embryos. Thus, the phiC31 system can be used in zebrafish for genetic manipulations, expanding the repertoire of available tools for genetic manipulation in this vertebrate model.
PMCID: PMC3019273  PMID: 20556509
Phic31 integrase; Site-specific recombination; Zebrafish
7.  Using retroviruses as a mutagenesis tool to explore the zebrafish genome 
We review different uses of the retroviral mutagenesis technology as the tool to manipulate the zebrafish genome. In addition to serving as a mutagen in a phenotype-driven forward mutagenesis screen as it was originally adapted for, retroviral insertional mutagenesis can also be exploited in reverse genetic approaches, delivering enhancer- and gene-trap vectors for the purpose of examining gene expression patterns and mutagenesis, making sensitized mutants amenable for chemical and genetic modifier screens, and producing gain-of-function mutations by epigenetically overexpressing the retroviral-inserted genes. From a technology point of view, we also summarize the recent advances in the high-throughput cloning of retroviral integration sites, a pivotal step toward identifying mutations. Lastly, we point to some potential directions that retroviral mutagenesis might take from the lessons of studying other model organisms.
PMCID: PMC2722255  PMID: 18977782
genetics; moloney murine leukemia virus; enhancer traps; gene traps; linker-mediated PCR; zebrafish

Results 1-7 (7)