Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

Nyabi, Omar; Naessens, Michael; Haigh, Katharina; Gembarska, Agnieszka; Goossens, Steven; Maetens, Marion; Clercq, Sarah De; Drogat, Benjamin; Haenebalcke, Lieven; Bartunkova, Sonia; Vos, Ilse De; Craene, Bram De; Karimi, Mansour; Berx, Geert; Nagy, András; Hilson, Pierre; Marine, Jean–Christophe; Haigh, Jody J.

doi:10.1093/nar/gkp112

Cited by 104 publications

(144 citation statements)

References 43 publications

(55 reference statements)

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“…Surviving clones were isolated and analyzed for homologous recombination by Southern blot using EcoRI and KpnI double-digested genomic DNA. Proper targeting was verified with 5′ and 3′ external probes [kindly provided by Christine Hartmann (Nyabi et al, 2009)]. Expression of the FLAG EnRCdx1-IRES-GFP bicistronic transcript after removal of the PGKpNeo stop cassette was verified by anti-FLAG western blotting analysis and GFP fluorescence following Cre transfection ( pMC-Cre) of one targeted ESC clone.…”

Section: Plasmid Constructsmentioning

confidence: 99%

A direct role for murine Cdx proteins in the trunk neural crest-gene regulatory network

et al. 2016

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Numerous studies in chordates and arthropods currently indicate that Cdx proteins have a major ancestral role in the organization of posthead tissues. In urochordate embryos, Cdx loss-of-function has been shown to impair axial elongation, neural tube (NT) closure and pigment cell development. Intriguingly, in contrast to axial elongation and NT closure, a Cdx role in neural crest (NC)-derived melanocyte/ pigment cell development has not been reported in any other chordate species. To address this, we generated a new conditional pan-Cdx functional knockdown mouse model that circumvents Cdx functional redundancy as well as the early embryonic lethality of Cdx mutants. Through directed inhibition in the neuroectoderm, we provide in vivo evidence that murine Cdx proteins impact melanocyte and enteric nervous system development by, at least in part, directly controlling the expression of the key early regulators of NC ontogenesis Pax3, Msx1 and Foxd3. Our work thus reveals a novel role for Cdx proteins at the top of the trunk NC gene regulatory network in the mouse, which appears to have been inherited from their ancestral ortholog.

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Section: Plasmid Constructsmentioning

confidence: 99%

A direct role for murine Cdx proteins in the trunk neural crest-gene regulatory network

et al. 2016

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“…In theory, a reporter line driven by the ROSA26 locus or a CAG promoter is ubiquitously expressed, and recent work has demonstrated that the CAG promoter is eight to tenfold stronger than the ROSA26 promoter (Nyabi et al 2009;Chen et al 2011). xA more complex reporter line that is commonly used is Z/EG.…”

Section: Reporter Linesmentioning

confidence: 99%

Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Cox

Liu

Lagarde

et al. 2012

JARO

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In recent years, there has been significant progress in the use of Cre-loxP technology for conditional gene expression in the inner ear. Here, we introduce the basic concepts of this powerful technology, emphasizing the differences between Cre and CreER. We describe the creation and Cre expression pattern of each Cre and CreER mouse line that has been reported to have expression in auditory and vestibular organs. We compare the Cre expression patterns between Atoh1-CreER TM and Atoh1-CreER T2 and report a new line, Fgfr3-iCreER T2 , which displays inducible Cre activity in cochlear supporting cells. We also explain how results can vary when transgenic vs. knock-in Cre/CreER alleles are used to alter gene expression. We discuss practical issues that arise when using the Cre-loxP system, such as the use of proper controls, Cre efficiency, reporter expression efficiency, and Cre leakiness. Finally, we introduce other methods for conditional gene expression, including Flp recombinase and the tetracycline-inducible system, which can be combined with Cre-loxP mouse models to investigate conditional expression of more than one gene.

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“…45,46 Melanin content determination. Melanin was extracted with an alkaline solution as previously described.…”

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confidence: 99%

Identification of a ZEB2-MITF-ZEB1 transcriptional network that controls melanogenesis and melanoma progression

et al. 2014

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Deregulation of signaling pathways that control differentiation, expansion and migration of neural crest-derived melanoblasts during normal development contributes also to melanoma progression and metastasis. Although several epithelial-tomesenchymal (EMT) transcription factors, such as zinc finger E-box binding protein 1 (ZEB1) and ZEB2, have been implicated in neural crest cell biology, little is known about their role in melanocyte homeostasis and melanoma. Here we show that mice lacking Zeb2 in the melanocyte lineage exhibit a melanoblast migration defect and, unexpectedly, a severe melanocyte differentiation defect. Loss of Zeb2 in the melanocyte lineage results in a downregulation of the Microphthalmia-associated transcription factor (Mitf) and melanocyte differentiation markers concomitant with an upregulation of Zeb1. We identify a transcriptional signaling network in which the EMT transcription factor ZEB2 regulates MITF levels to control melanocyte differentiation. Moreover, our data are also relevant for human melanomagenesis as loss of ZEB2 expression is associated with reduced patient survival. Melanocytes are specialized cells in the skin that produce melanin, a pigment that is responsible for skin and hair color and that provides protection against ultraviolet (UV) radiation. During mouse embryogenesis, melanoblasts originate from the neural crest and migrate along a dorsolateral pathway from the neural tube to the developing dermis.1 Around embryonic day (E) E11 they move into the epidermis and eventually populate the developing hair follicle.2 Here they separate into two distinct populations: the differentiated pigmented melanocytes, which reside in the hair matrix, and the non-pigmented melanocyte stem cells (MSC) in the bulge. The latter cells are responsible for replenishing the hair follicle with new melanocytes during each hair cycle. Genetic studies in mice demonstrated the importance of several key players (such as sex-determining region Y (SRY)-box 10 (Sox10), paired-box 3 (Pax3), microphthalmia-associated transcription factor (Mitf), endothelin 3/endothelin receptor B (Edn3/ Ednrb), Kitl/Kit, Slug, cellular myelocytomatosis oncogene cellular homolog (cMyc) and b-Catenin (b-Cat)) for melanoblast cell fate specification, proliferation, migration and survival.2-4 The master regulator of the melanocyte development is MITF, which is spatio-temporally controlled by several key transcription factors such as SOX10, PAX3 and b-catenin.5-7 Fundamentally, MITF induces gene expression patterns that prompt a melanocyte to differentiate and initiate pigment production by activating genes important for melanin biosynthesis (such as Tyrosinase (Tyr), Dopachrome tautomerase (Dct), Tyrosinase-related protein 1 (Tyrp1) and

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Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

Cited by 104 publications

References 43 publications

A direct role for murine Cdx proteins in the trunk neural crest-gene regulatory network

A direct role for murine Cdx proteins in the trunk neural crest-gene regulatory network

Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Identification of a ZEB2-MITF-ZEB1 transcriptional network that controls melanogenesis and melanoma progression

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