Article

Efficient shRNA-Mediated Inhibition of Gene Expression in Zebrafish

1 Whitehead Institute for Biomedical Research , Cambridge Massachusetts.
Zebrafish (Impact Factor: 1.77). 07/2012; 9(3):97-107. DOI: 10.1089/zeb.2012.0770
Source: PubMed

ABSTRACT Abstract Despite the broad repertoire of loss of function (LOF) tools available for use in the zebrafish, there remains a need for a simple and rapid method that can inhibit expression of genes at later stages. RNAi would fulfill that role, and a previous report (Dong et al. 2009) provided encouraging data. The goal of this study was to further address the ability of expressed shRNAs to inhibit gene expression. This included quantifying RNA knockdown, testing specificity of shRNA effects, and determining whether tissue-specific LOF could be achieved. Using an F0 transgenic approach, this report demonstrates that for two genes, wnt5b and zDisc1, each with described mutant and morphant phenotypes, shRNAs efficiently decrease endogenous RNA levels. Phenotypes elicited by shRNA resemble those of mutants and morphants, and are reversed by expression of cognate RNA, further demonstrating specificity. Tissue-specific expression of zDisc1 shRNAs in F0 transgenics demonstrates that conditional LOF can be readily obtained. These results suggest that shRNA expression presents a viable approach for rapid inhibition of zebrafish gene expression.

1 Follower
 · 
102 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Clonal evolution is the process by which genetic and epigenetic diversity is created within malignant tumor cells. This process culminates in a heterogeneous tumor, consisting of multiple subpopulations of cancer cells that often do not contain the same underlying mutations. Continuous selective pressure permits outgrowth of clones that harbor lesions that are capable of enhancing disease progression, including those that contribute to therapy resistance, metastasis and relapse. Clonal evolution and the resulting intratumoral heterogeneity pose a substantial challenge to biomarker identification, personalized cancer therapies and the discovery of underlying driver mutations in cancer. The purpose of this Review is to highlight the unique strengths of zebrafish cancer models in assessing the roles that intratumoral heterogeneity and clonal evolution play in cancer, including transgenesis, imaging technologies, high-throughput cell transplantation approaches and in vivo single-cell functional assays.
    Disease Models and Mechanisms 07/2014; 7(7):755-762. DOI:10.1242/dmm.015842 · 5.54 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The Japanese medaka, Oryzias latipes, is a vertebrate teleost model with a long history of genetic research. A number of unique features and established resources distinguish medaka from other vertebrate model systems. A large number of laboratory strains from different locations are available. Due to a high tolerance to inbreeding, many highly inbred strains have been established, thus providing a rich resource for genetic studies. Furthermore, closely related species native to different habitats in Southeast Asia permit comparative evolutionary studies. The transparency of embryos, larvae, and juveniles allows a detailed in vivo analysis of development. New tools to study diverse aspects of medaka biology are constantly being generated. Thus, medaka has become an important vertebrate model organism to study development, behavior, and physiology. In this review, we provide a comprehensive overview of established genetic and molecular-genetic tools that render medaka fish a full-fledged vertebrate system. M EDAKA (Oryzias latipes) is a small freshwater fish of the family Adrianichthyidae in the order Beloni-formes. It is closely related to other members of the super-order Acanthopterygii of ray-finned fish such as pufferfish (tetraodon and fugu), stickleback and killifish, while it is separated from zebrafish by 150 million years (MY) of divergent evolution (Figure 1). A growing number of se-quenced teleost genomes and several established genetic model systems (zebrafish, stickleback, medaka) thus provide a unique resource for comparative studies relating to vertebrate evolution. Medaka is native to Taiwan, Korea, China, and Japan. In Japan it is found in small rivers, creeks, and rice paddies on all main islands with the exception of Hok-kaido. Medaka is a euryhaline species and can also live in brackish water (Figure 2, A and B) (Inoue and Takei 2002). As a resident of a temperate zone, medaka can tolerate a wide range of temperatures (4–40°) both as adult and embryo (Sampetrean et al. 2009). Thus, in the laboratory, temperature can be used to control the developmental speed without adverse effects. Medaka native to Japan and East Korea have a diploid karyotype of 48 chromosomes, but 46 chromosomes in medaka from West Korea and China (Uwa and Ojima 1981). The different chromosome number is due to a fusion of chromosome 11 and 13 by a Robertsonian translocation (Myosho et al. 2012b). The haploid genome size is 800 Mb (Kasahara et al. 2007). Adults can reach a length of up to 4 cm and the wild-type pigmentation is greyish-brown (Figure 2D). However, mutant strains are available where body pigmentation is strongly reduced both at embryonic and adult stages (Figure 2C) (Wakamatsu et al. 2001; Kelsh et al. 2004). Interestingly, sex-specific pigmentation can be used to distinguish male from female embryos as early as 3 days postfertilization (dpf) (organogenesis stages) (Wada et al. 1998), since the leucophore free (lf, slc2a15b) locus that is required for pigmentation by leucophores is located on the sex chromosomes X and Y (Kimura et al. 2014). The XX–XY sex determination system (see below) allows the establishment of mutant strains with females (XX) that are homozy-gous for a loss-of-function lf allele and males (XY) that are heterozygous and will therefore exhibit wild-type leucophore pigmentation. The strains, Qurt and FLF2, have the wild allele
    Genetics 04/2015; 199(4):905-918. DOI:10.1534/genetics.114.173849 · 4.87 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Precise vectorial transport of rhodopsin is essential for rod photoreceptor health and function. Mutations that truncate or extend the C terminus of rhodopsin disrupt this transport, and lead to retinal degeneration and blindness in human patients and in mouse models. Here we show that such mutations disrupt the binding of rhodopsin to the small GTPase rab11a. The rhodopsin-rab11a interaction is a direct binding interaction that does not depend on the nucleotide binding state of rab11a. Expression of EGFP-rab11a fusion proteins in Xenopus laevis photoreceptors revealed that the nucleotide binding status of rab11a affects its subcellular localization, with GTP-locked mutants concentrated in the inner segment and GDP-locked mutants concentrated in the outer segment. shRNA-mediated knockdown of rab11a in rods led to shortened outer segments and retinal degeneration. Together, our results show the critical importance of direct rhodopsin-rab11a interactions for the formation and maintenance of vertebrate photoreceptors.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 11/2014; 34(45):14854-63. DOI:10.1523/JNEUROSCI.1943-14.2014 · 6.75 Impact Factor

Preview

Download
7 Downloads
Available from