Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity

Sakuma, Tetsushi; Ochiai, Hiroshi; Kaneko, Takehito; Mashimo, Tomoji; Tokumasu, Daisuke; Sakane, Yuto; Suzuki, Ken; Miyamoto, Tatsuo; Sakamoto, Naoaki; Matsuura, Shinya; Yamamoto, Takashi

doi:10.1038/srep03379

Cited by 208 publications

(183 citation statements)

References 28 publications

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“…Platinum Gate TALEN Kit (Addgene; Kit #1000000043) was used to construct a TALEN expression vector as described previously. 50 A CAG promoter-driven vector was used as the destination vector. To evaluate mutagenic efficiency, NPHS1-TALENs were transfected into HEK293 cells using Lipofectamine 2000 (Life Technologies).…”

Section: Generation Of Nphs1-talen Plasmids and Validation Of Their Amentioning

confidence: 99%

Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation

Sharmin¹,

Taguchi²,

Kaku³

et al. 2015

JASN

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202

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Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator-like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro. These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm-like structures. Microarray analysis of sorted iPS cell-derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo. Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell-derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell-derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases.

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Section: Generation Of Nphs1-talen Plasmids and Validation Of Their Amentioning

confidence: 99%

Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation

Sharmin¹,

Taguchi²,

Kaku³

et al. 2015

JASN

Self Cite

202

214

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“…The N-and C-terminal domains of TALE and the FokI nuclease domain were taken from the Platinum Gate TALEN Kit (Sakuma et al, 2013). Epib expression was similarly downregulated in experimental embryos (Fig.…”

Section: Gene Knockdown and Knockoutmentioning

confidence: 99%

Tfap2 and Sox1/2/3 cooperatively specify ectodermal fates in ascidian embryos

et al. 2016

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Epidermis and neural tissues differentiate from the ectoderm in animal embryos. Although epidermal fate is thought to be induced in vertebrate embryos, embryological evidence has indicated that no intercellular interactions during early stages are required for epidermal fate in ascidian embryos. To test this hypothesis, we determined the gene regulatory circuits for epidermal and neural specification in the ascidian embryo. These circuits started with Tfap2-r.b and Sox1/2/3, which are expressed in the ectodermal lineage immediately after zygotic genome activation. Tfap2-r.b expression was diminished in the neural lineages upon activation of fibroblast growth factor signaling, which is known to induce neural fate, and sustained only in the epidermal lineage. Tfap2-r. b specified the epidermal fate cooperatively with Dlx

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“…When a TALEN induces a double-strand break (DSB) a mutation can occur when the cellular DNA repair mechanisms fail (Cermak et al, 2011). Recently, a range of improvements has been made to the basic TALEN architecture to improve the mutational efficiency, mostly by reducing the length of the N-and C-terminal regions (Sakuma et al, 2013a;Sakuma et al, 2013b;Bedell et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function inCiona

et al. 2014

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Custom designed nucleases can simplify gene targeting experiments and have the potential to allow these techniques to be performed in a wide range of organisms. Transcriptional activator-like effector nucleases (TALENs) are starting to fulfill this potential with the advantages of low cost and fast construction times. Here, we report that TALENs are highly effective at inducing mutations in specific genomic loci in the ascidian chordate Ciona intestinalis. In Ciona there are well-established methods to introduce exogenous DNA by electroporation, and we show that this method can be used to introduce constructs that can express TALENs ubiquitously or in specific tissues. Our current protocols enable the rapid analysis of hundreds of TALEN-induced mutants. TALEN electroporations result in a high rate of mutations. These mutations can result in gene knockouts that recapitulate previously described functions of Fgf3 and Hox12. We show that TALENs can work efficiently to cause tissue-specific knockouts and demonstrate this by knocking out Hox12 in the epidermis and Fgf3 in neural tissues. We also use tissue-specific knockouts to reveal a new function of Fgf3 during ascidian larval metamorphosis.

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Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity

Cited by 208 publications

References 28 publications

Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation

Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation

Tfap2 and Sox1/2/3 cooperatively specify ectodermal fates in ascidian embryos

Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function inCiona

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