Abstract
BackgroundReduced expression of developmentally important genes and tumor suppressors due to haploinsufficiency or epigenetic suppression has been shown to contribute to the pathogenesis of various malignancies. However, methodology that allows spatio-temporally knockdown of gene expression in various model organisms such as zebrafish has not been well established, which largely limits the potential of zebrafish as a vertebrate model of human malignant disorders.Principal FindingHere, we report that multiple copies of small hairpin RNA (shRNA) are expressed from a single transcript that mimics the natural microRNA-30e precursor (mir-shRNA). The mir-shRNA, when microinjected into zebrafish embryos, induced an efficient knockdown of two developmentally essential genes chordin and α-catenin in a dose-controllable fashion. Furthermore, we designed a novel cassette vector to simultaneously express an intronic mir-shRNA and a chimeric red fluorescent protein driven by lineage-specific promoter, which efficiently reduced the expression of a chromosomally integrated reporter gene and an endogenously expressed gata-1 gene in the developing erythroid progenitors and hemangioblasts, respectively.SignificanceThis methodology provides an invaluable tool to knockdown developmental important genes in a tissue-specific manner or to establish animal models, in which the gene dosage is critically important in the pathogenesis of human disorders. The strategy should be also applicable to other model organisms.
Highlights
Understanding the development and disease-associated molecular and cellular processes in model organisms has largely relied on gene loss-of-function approaches
These data suggest that a narrow window of reduced expression of a tumor suppressor is crucial for acute myeloid leukemia and solid tumor development
Efficient Knockdown of Reporter Gene In Vivo by MirshRNA It has been previously shown that the 59 and 39 flanking sequences of miRNA precursor are crucial for miRNA processing and maturation [16], and the hairpin small hairpin RNA (shRNA) can be expressed from a synthetic stem-loop precursor flanked by the 59 and 39 flanking sequences of either human miR-30 [14] or mouse miR155 gene [13]
Summary
Understanding the development and disease-associated molecular and cellular processes in model organisms has largely relied on gene loss-of-function approaches. Heritable targeted gene disruption with designed zinc-finger nucleases has been reported to inactivate zebrafish golden, ntl and vascular endothelial growth factor-2 receptor genes [2,3] While these technologies will undoubtedly speed up the dissection of signal transduction pathways/networks during development and evolution, a conditional knockdown system with stably titering down gene dosage in a tissue-specific fashion is unavailable. This latter strategy is critically important toward establishing zebrafish as a vertebrate model of human pathological conditions and diseases [4], because increasing evidence has demonstrated that haploinsufficiency and epigenetic suppression of tumor suppressor genes, other than complete mutational inactivation or permanent removal of genetic material from the host genome, might be a preferred mechanism in promoting cell transformation [5]. The strategy should be applicable to other model organisms
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