Floxin, a resource for genetically engineering mouse ESCs

Singla, Veena; Hunkapiller, Julie; Santos, Nicole; Seol, Allen D.; Norman, Andrew R.; Wakenight, Paul; Skarnes, William C.; Reiter, Jeremy F.

doi:10.1038/nmeth.1406

Cited by 26 publications

(38 citation statements)

References 17 publications

(16 reference statements)

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“…To study how misactivation of GLI2 causes cancer, we used the Floxin system to generate a Gli2-knockin allele that encodes a fusion of GLI2 to EGFP and FLAG tags (Gli2-EGFP) (7). Mice homozygous for the Gli2-EGFP allele are viable and morphologically indistinguishable from WT, revealing that this fusion protein is functional (8).…”

Section: Resultsmentioning

confidence: 99%

Hedgehog signaling drives medulloblastoma growth via CDK6

Raleigh¹,

Choksi

Krup

et al. 2017

Journal of Clinical Investigation

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Section: Resultsmentioning

confidence: 99%

Hedgehog signaling drives medulloblastoma growth via CDK6

Raleigh¹,

Choksi

Krup

et al. 2017

Journal of Clinical Investigation

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“…Independently derived lines of ROSA26CreER;Chd1 fl/− ESCs were used for all in vitro analyses in this work, with the exception of the microarray studies and colony formation assays. These were carried out using Chd1 −/− ESCs, derived by sequential targeting of Chd1 +/− ESCs with the construct described above, and the Chd1-Flag ChIP-qPCR, which was carried out using a Chd1 gene trap ES cell line (RRF067), in which a Flag epitope was knocked in at the Chd1 locus by floxin technology (Singla et al, 2010).…”

Section: Esc Derivationmentioning

confidence: 99%

Chd1 is essential for the high transcriptional output and rapid growth of the mouse epiblast

et al. 2015

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The pluripotent mammalian epiblast undergoes unusually fast cell proliferation. This rapid growth is expected to generate a high transcriptional demand, but the underlying mechanisms remain unknown. We show here that the chromatin remodeler Chd1 is required for transcriptional output and development of the mouse epiblast. Chd1 −/− embryos exhibit proliferation defects and increased apoptosis, are smaller than controls by E5.5 and fail to grow, to become patterned or to gastrulate. Removal of p53 allows progression of Chd1 −/− mutants only to E7.0-8.0, highlighting the crucial requirement for Chd1 during early post-implantation development. Chd1embryonic stem cells (ESCs) have a self-renewal defect and a genome-wide reduction in transcriptional output at both known mRNAs and intergenic transcripts. These transcriptional defects were only uncovered when cell number-normalized approaches were used, and correlate with a lower engagement of RNAP II with transcribed genes in Chd1 −/− ESCs. We further show that Chd1 directly binds to ribosomal DNA, and that both Chd1 −/− epiblast cells in vivo and ESCs in vitro express significantly lower levels of ribosomal RNA. In agreement with these findings, mutant cells in vivo and in vitro exhibit smaller and more elongated nucleoli. Thus, the RNA output by both Pol I and II is reduced in Chd1 −/− cells. Our data indicate that Chd1 promotes a globally elevated transcriptional output required to sustain the distinctly rapid growth of the mouse epiblast.

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“…Electroporation for reversion and Floxin were performed as described previously (Singla et al, 2010). Floxin clones were selected with 150 mg/ml G418 and were assessed by genomic PCR and b-galactosidase activity using the Galacto-Light Plus System (Applied Biosystems).…”

Section: Electroporation Selection and Screeningmentioning

confidence: 99%

“…In this study, we used an efficient knock-in approach called the Floxin system to genetically tailor the Gli2 locus in mouse embryonic stem cells (ESCs) (Singla et al, 2010). We created a panel of GFP-tagged Gli2 alleles that systematically delete or modify domains or residues implicated in Gli2 function.…”

Section: Introductionmentioning

confidence: 99%

A central region of Gli2 regulates its localization to the primary cilium and transcriptional activity

Santos

Reiter

2014

Journal of Cell Science

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Signaling through vertebrate Hedgehog (Hh) proteins depends on the primary cilium. In response to Hh signals, the transcriptional activator of the pathway, Gli2, accumulates at the ciliary tip, raising the possibility that ciliary localization is important for Gli2 activation. To test this hypothesis, we used the Floxin system to create knockin Gli2 alleles in embryonic stem cells (ESCs) to allow methodical testing of which domains and residues are essential for the ciliary localization of Gli2. The Gli2 zinc fingers, transcriptional activation domain, repressor domain, phosphorylation cluster and a Sufu binding motif were each dispensable for ciliary localization. Mutating residues that are required for Gli2 sumoylation and nuclear trafficking also did not abrogate ciliary localization. By contrast, several other domains restricted Gli2 nuclear localization, and a central region, distinct from previously characterized domains, was required for ciliary localization. In addition to an inability to localize to cilia, Gli2 lacking this central domain was unable to activate target genes. Thus, our systematic analysis in ESCs reveals that distinct regions of Gli2 regulate its nuclear and ciliary localization. The identification of a domain essential for both ciliary localization and transcriptional activity suggests that ciliary localization of Gli2 is required for its activation.

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Floxin, a resource for genetically engineering mouse ESCs

Cited by 26 publications

References 17 publications

Hedgehog signaling drives medulloblastoma growth via CDK6

Hedgehog signaling drives medulloblastoma growth via CDK6

Chd1 is essential for the high transcriptional output and rapid growth of the mouse epiblast

A central region of Gli2 regulates its localization to the primary cilium and transcriptional activity

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