Off-Target Effect of doublecortin Family shRNA on Neuronal Migration Associated with Endogenous MicroRNA Dysregulation
Seung Tae BaekGéraldine KerjanStephanie BielasJi Eun LeeAli G. FenstermakerGaia NovarinoJoseph G. Gleeson
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Doublecortin
Gene knockout
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Ribonuclease III
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The widespread use of gene knockout technology promotes the course of the research of gene function in post-genomic era greatly. Recently, the research of p38#alpha# knockout mice, which is one member of MAPKs, is very typical in filtering noise from critical signals in signal transduction and translating phenotype into gene function. Looking back on the research of p38#alpha# knockout mice showed: the reasonable use of gene knockout technology, based on carefully designed and analyzed mice, is very important for defining the function in vivo of important signal molecular and pushing forward the research of function gene.
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The widespread use of gene knockout technology promotes the course of the research of gene function in post genomic era greatly. Recently, the research of p 38 α knockout mice, which is one member of MAPKs, is very typical in filtering noise from critical signals in signal transduction and translating phenotype into gene function. Looking back on the research of p 38 α knockout mice showed: the reasonable use of gene knockout technology, based on carefully designed and analyzed mutant mice, is very important for defining the function in vivo of important signal molecular and pushing forward the research of function gene.
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Conditional gene knockout
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Knockout mice are useful tools that can provide information about the normal function of genes, including their biochemical, developmental, and physiological roles. One problem associated with the generation of knockout mice is that the loss of some genes of interest produces a lethal phenotype. Therefore, the use of conditioned knockout mice, in which genes are disrupted in specific organs, is essential for the elucidation of disease pathogenesis and the verification of drug targets. In general, conditional knockout mice are produced using the Cre/loxP system; however, the production of the large numbers of Cre/flox knockout and control mice required for analysis requires substantial time and effort. Here, we describe the generation of liver-specific conditional knockout mice via the introduction of lipid nanoparticles encapsulating Cre mRNA into the liver of floxed mice. This technique does not require the production of offspring by mating floxed mice and is therefore more convenient than the conventional method. The results presented here demonstrate that the LNP-based method enables liver-specific gene knockout in a short period of time.
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Gene knockout mice are desirable animal model for researching the function of gene and protein in vitro. There are some disadvantages in traditional knockout technology, such as complicated analysis of phenotype, long cycle time, numerous operations, etc, so the key problem to apply this technology better to medical research at present is how to set up knockout mice model more scientifically and more conveniently. This review introduced the latest advances in the research of gene knockout mice on several aspects, which included genetic background of embryonic stem cell, tissue-specific gene expression and innovation of gene targeting technology. [
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Doublecortin
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Analyses of mice carrying a deletion of the pan-neurotrophin receptor p75NTR have allowed identifying p75NTR as an important structural regulator of the hippocampus. Most of the previous analyses were done using p75NTR (ExIII) knockout mice which still express the short isoform of p75NTR. To scrutinize the role of p75NTR in the hippocampus, we analyzed adult and aged p75NTR (ExIV) knockout mice, in which both, the short and the full-length isoform are deleted. Deletion of these isoforms induced morphological alterations in the adult dentate gyrus (DG), leading to an increase in the thickness of the molecular and granular layer. Based on these observations, we next determined the morphological substrates that might contribute to this phenotype. The cholinergic innervation of the molecular and granular layer of the DG was found to be significantly increased in the knockout mice. Furthermore, adult neurogenesis in the DG was found to be significantly altered with increased numbers of doublecortin (DCX) positive cells and reduced numbers of apoptotic cells in p75NTR (ExIV) knockout mice. However, cell proliferation as measured by phosphohiston H3 (PH3) positive cell numbers was not affected. These morphological alterations (number of DCX-positive cells and increased cholinergic fiber densities) as well as reduced cell death in the DG are likely to contribute to the observed thickening of the granular layer in p75NTR (ExIV) knockout mice. In addition, Sholl-analysis of DCX-positive neurons revealed a higher dendritic complexity and could thus be a possible morphological correlate for the increased thickness of the molecular layer in p75NTR deficient animals. Our data clearly demonstrate that deletion of both, the short and the full-length isoform of p75NTR affects DG morphology, due to alterations of the cholinergic system and an imbalance between neurogenesis and programmed cell death within the subgranular zone.
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RNAi by short hairpin RNA (shRNA) is a powerful tool not only for studying gene functions in various organisms, including mammals, but also for the treatment of severe disorders. However, shRNA-expressing vectors can induce type I interferon (IFN) expression by activation of innate immune responses, leading to off-target effects and unexpected side effects. Several strategies have been developed to prevent type I IFN induction. On the other hand, it has remained unclear whether type I IFNs have effects on shRNA-mediated RNAi. Here, we show that the type I IFNs significantly inhibit shRNA-mediated RNAi. Treatment with recombinant human IFN-α significantly inhibited shRNA-mediated knockdown of target genes, while it did not inhibit small interfering RNA (siRNA)-mediated knockdown. Following treatment with IFN-α, increased and decreased copy numbers of shRNA and its processed form, respectively, were found in the cells transfected with shRNA-expressing plasmids. Dicer protein levels were not altered by IFN-α. These results indicate that type I IFNs inhibit shRNA-mediated RNAi via inhibition of dicer-mediated processing of shRNA to siRNA. Our findings should provide important clues for efficient RNAi-mediated knockdown of target genes in both basic researches and clinical gene therapy.
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Gene knockouts in mice have been reported for the glucocorti-coid, estrogen, and progesterone receptors, and a natural gene knockout, the testicular-feminized mouse, exists for the androgen receptor. Of these knockouts only the glucocorticoid receptor knockout has profound effects on embryonic development, with defects in the lung and adrenal gland causing perinatal lethality. Female mice with either the estrogen or progesterone receptor knocked out are infertile, as are male mice lacking the estrogen receptor, which have smaller testes and lower sperm production. Estrogen receptor knockout mice also suffer osteoporosis. Progesterone receptor knockout mice fail to display ovarian follicular rupture and normal sexual behavior. Steroid receptor knockout mice thus provide a useful animal model for further studies on steroid action. Recent results with Cre- and FLP-recombinase promise a new generation of gene-targeted mice by production of tissue-specific and ligand-inducible gene knockouts.
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