Mammalian Reverse Genetics without Crossing Reveals Nr3a as a Short-Sleeper Gene

Sunagawa, Genshiro A.; Sumiyama, Kenta; Ukai-Tadenuma, Maki; Perrin, Dimitri; Fujishima, Hiroshi; Ukai, Hideki; Nishimura, Osamu; Shi, Shoi; Ohno, Rei-ichiro; Narumi, Ryohei; Shimizu, Yoshihiro; Tone, Daisuke; Ode, Koji L.; Kuraku, Shigehiro; Ueda, Hiroki R.

doi:10.1016/j.celrep.2015.12.052

Cited by 117 publications

(180 citation statements)

References 45 publications

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“…The detectable presence of dSTIM transcripts and protein in larval brain lysates of Elav C155 GAL4 > STIM dual ;cas9 suggests that a complete KO of both alleles may not occur in all neuronal cells marked by Elav C155 GAL4 , though some of the residual transcripts and protein could arise from nonneuronal cells, such as glia that do not express Elav C155 GAL4 . Recent studies suggest that increasing the target sites to three or more within a locus is a more dependable strategy for obtaining tissue-specific KOs (Sunagawa et al 2016). …”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas-Induced Mutants Identify a Requirement for dSTIM in Larval Dopaminergic Cells of Drosophila melanogaster

Pathak

Trivedi

Hasan

2017

G3 Genes|Genomes|Genetics

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Molecular components of store-operated calcium entry have been identified in the recent past and consist of the endoplasmic reticulum (ER) membrane-resident calcium sensor STIM and the plasma membrane-localized calcium channel Orai. The physiological function of STIM and Orai is best defined in vertebrate immune cells. However, genetic studies with RNAi strains in Drosophila suggest a role in neuronal development and function. We generated a CRISPR-Cas-mediated deletion for the gene encoding STIM in Drosophila (dSTIM), which we demonstrate is larval lethal. To study STIM function in neurons, we merged the CRISPR-Cas9 method with the UAS-GAL4 system to generate either tissue- or cell type-specific inducible STIM knockouts (KOs). Our data identify an essential role for STIM in larval dopaminergic cells. The molecular basis for this cell-specific requirement needs further investigation.

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Section: Discussionmentioning

confidence: 99%

CRISPR-Cas-Induced Mutants Identify a Requirement for dSTIM in Larval Dopaminergic Cells of Drosophila melanogaster

Pathak

Trivedi

Hasan

2017

G3 Genes|Genomes|Genetics

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“…Tamoxifen administration in mice was performed as previously described (Matsumoto et al, 2016). Mark1 mutant mice were generated using the triple CRISPR method as previously described (Sunagawa et al, 2016).…”

Section: Micementioning

confidence: 99%

“…To examine the role of MARK1 in vivo, mutant animals were generated using the triple CRISPR method (Sunagawa et al, 2016). Synthesized Cas9 mRNA and guide RNAs (gRNAs) were microinjected into cytoplasm of B6C3F1 one-cell embryos (Fig.…”

mentioning

confidence: 99%

Modulation of apical constriction by Wnt signaling is required for lung epithelial shape transition

Fumoto

Takigawa-Imamura

Sumiyama³

et al. 2016

Development

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In lung development, the apically constricted columnar epithelium forms numerous buds during the pseudoglandular stage. Subsequently, these epithelial cells change shape into the flat or cuboidal pneumocytes that form the air sacs during the canalicular and saccular (canalicular-saccular) stages, yet the impact of cell shape on tissue morphogenesis remains unclear. Here, we show that the expression of Wnt components is decreased in the canalicularsaccular stages, and that genetically constitutive activation of Wnt signaling impairs air sac formation by inducing apical constriction in the epithelium as seen in the pseudoglandular stage. Organ culture models also demonstrate that Wnt signaling induces apical constriction through apical actomyosin cytoskeletal organization. Mathematical modeling reveals that apical constriction induces bud formation and that loss of apical constriction is required for the formation of an air sac-like structure. We identify MAP/microtubule affinity-regulating kinase 1 (Mark1) as a downstream molecule of Wnt signaling and show that it is required for apical cytoskeletal organization and bud formation. These results suggest that Wnt signaling is required for bud formation by inducing apical constriction during the pseudoglandular stage, whereas loss of Wnt signaling is necessary for air sac formation in the canalicular-saccular stages.

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“…The NHEJ-dependent gene KO works stably in mammalian cultured cells, 28, 30, 56 ESCs and other pluripotent cells, or even mammalian zygotes. 57–59 Furthermore, simultaneous use of multiple sgRNAs can introduce mutations in multiple genes and create a large deletion between the targeted loci, as well as increase the KO efficiency 43, 59–62 (Fig. 2).…”

Section: Current Technologies For Efficient Genome Editing By Site-spmentioning

confidence: 99%

“…The actual rate reached to over 95% due to long deletions between CRISPR targeted sites. 62 A set of triple sgRNAs which cover ~80% of all genes in the mouse genome has been created as an open database (http://crispr.riken.jp/). Potential off-target effects can be also excluded by using the second set of triple sgRNAs that covers ~70% of all mouse genes.…”

Section: Current Technologies For Efficient Genome Editing By Site-spmentioning

confidence: 99%

Next-generation mammalian genetics toward organism-level systems biology

Susaki

Ukai²,

Ueda

2017

npj Syst Biol Appl

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Organism-level systems biology in mammals aims to identify, analyze, control, and design molecular and cellular networks executing various biological functions in mammals. In particular, system-level identification and analysis of molecular and cellular networks can be accelerated by next-generation mammalian genetics. Mammalian genetics without crossing, where all production and phenotyping studies of genome-edited animals are completed within a single generation drastically reduce the time, space, and effort of conducting the systems research. Next-generation mammalian genetics is based on recent technological advancements in genome editing and developmental engineering. The process begins with introduction of double-strand breaks into genomic DNA by using site-specific endonucleases, which results in highly efficient genome editing in mammalian zygotes or embryonic stem cells. By using nuclease-mediated genome editing in zygotes, or ~100% embryonic stem cell-derived mouse technology, whole-body knock-out and knock-in mice can be produced within a single generation. These emerging technologies allow us to produce multiple knock-out or knock-in strains in high-throughput manner. In this review, we discuss the basic concepts and related technologies as well as current challenges and future opportunities for next-generation mammalian genetics in organism-level systems biology.

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Mammalian Reverse Genetics without Crossing Reveals Nr3a as a Short-Sleeper Gene

Cited by 117 publications

References 45 publications

CRISPR-Cas-Induced Mutants Identify a Requirement for dSTIM in Larval Dopaminergic Cells of Drosophila melanogaster

CRISPR-Cas-Induced Mutants Identify a Requirement for dSTIM in Larval Dopaminergic Cells of Drosophila melanogaster

Modulation of apical constriction by Wnt signaling is required for lung epithelial shape transition

Next-generation mammalian genetics toward organism-level systems biology

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