Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei

Prigge, Justin R.; Wiley, James; Talago, Emily A.; Young, Elise M.; Johns, Laura; Kundert, Jean A.; Sonsteng, Katherine M.; Halford, William P.; Capecchi, Mario R.; Schmidt, Edward E.

doi:10.1007/s00335-013-9469-8

Cited by 70 publications

(89 citation statements)

References 43 publications

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“…ROSA nT-nG mice contain a fluorescent allele that expresses nuclear-localized red fluorescence (tdTomato) in all cells, except those exposed to Cre recombinase; the latter cells express nuclear-localized enhanced green fluorescent protein (EGFP) (7). In ROSA nT-nG , Pax6α::Cre flat-mounted retinas and sections from 3-mo-old mice, Crerecombined cells (green) were detected predominantly in the distal retina ( Fig.…”

Section: Significancementioning

confidence: 99%

Genetic rescue models refute nonautonomous rod cell death in retinitis pigmentosa

Koch

Duong

Hsu

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Fig. 3. Restoration of PDE6b expression rescues rods and partially restores OS length, in a dose-dependent fashion, and prevents cone death. Pde6b STOP / Pde6b H620Q , Pde6g::CreERT2 mice were injected with 25, 50, or 100 μg/g BW tamoxifen to rescue 30% (T30), 50% (T50), or 70% (T70) of rods, respectively; untreated mutants (mut) and wild-type mice (Pde6b + /Pde6b H620Q , Pde6g::CreERT2, WT) were not injected. Tamoxifen injections were in 1-mo-old mice; at 9 mo of age, retinas were sectioned and immunolabeled. (A) Antibodies: rhodopsin or PDE6b (rod OSs, red) and arrestin (cones, green). Hoechst dye, nuclei, blue. Arrows, arrestin-immunopositive cone cell bodies; arrowheads, arrestin-immunopositive cone OSs. (B) Rhodopsin and arrestin immunolabeled sections were quantitatively analyzed for ONL thickness, cone number, rod OS length and cone IS + OS length. Gray dots, individual mice (n = 4 for each group); horizontal black lines, group means. Treated and untreated groups were compared by a linear regression model: ***P < 0.001. IS, inner segment; ONL, outer nuclear layer; OS, outer segment. (Scale bar, 15 μm.)

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Section: Significancementioning

confidence: 99%

Genetic rescue models refute nonautonomous rod cell death in retinitis pigmentosa

Koch

Duong

Hsu

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…In the absence of Cre activity, ROSA mT−mG/mT−mG or ROSA nT−nG/nT−nG mice exhibit no EGFP expression in any tissues including liver [2], [3], [15], [30]–[32]. Hydrodynamic delivery of 25 µg of pMC1-Cre plasmid, which uses a very weak ubiquitously active promoter [28] to drive Cre expression, into ROSA mT−mG/mT−mG mice resulted in ∼3%–10% of the hepatocytes stably converting to green, with preferential targeting of mid-zone hepatocytes (Fig.…”

Section: Resultsmentioning

confidence: 99%

Hydrodynamic Delivery of Cre Protein to Lineage-Mark or Time-Stamp Mouse Hepatocytes In situ

et al. 2014

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Cre-responsive fluorescent marker alleles are powerful tools for cell lineage tracing in mice; however their utility is limited by regulation of Cre activity. When targeting hepatocytes, hydrodynamic delivery of a Cre-expression plasmid can convert Cre-responsive alleles without inducing the intracellular or systemic antiviral responses often associated with viral-derived Cre-expression vectors. In this method, rapid high-volume intravenous inoculation induces hepatocyte-targeted uptake of extracellular molecules. Here we tested whether hydrodynamic delivery of Cre protein or Cre fused to the HIV-TAT cell-penetrating peptide could convert Cre-responsive reporters in hepatocytes of mice. Hydrodynamic delivery of 2 nmol of either Cre or TAT-Cre protein converted the reporter allele in 5 to 20% of hepatocytes. Neither protein gave detectable Cre activity in endothelia, non-liver organs, or non-hepatocyte cells in liver. Using mice homozygous for a Cre-responsive marker that directs red- (Cre-naïve) or green- (Cre-converted) fluorescent proteins to the nucleus, we assessed sub-saturation Cre-activity. One month after hydrodynamic inoculation with Cre protein, 58% of hepatocyte nuclei that were green were also red, indicating that less than half of the hepatocytes that had obtained enough Cre to convert one marker allele to green were able to convert all alleles. For comparison, one month after hydrodynamic delivery of a Cre-expression plasmid with a weak promoter, only 26% of the green nuclei were also red. Our results show that hydrodynamic delivery of Cre protein allows rapid allelic conversion in hepatocytes, but Cre-activity is sub-saturating so many cells will not convert multiple Cre-responsive alleles.

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“…S4B). Although Hopx CreER can label AT2 cells (Jain et al, 2015), such leaky targeting was infrequent when induced at E19 using the nuclear reporter Rosa nTnG (Prigge et al, 2013) (7/189 GFP + cells from three mice; Fig. 3A), suggesting that Hopx…”

Section: At1 Cells Fold In Conjunction With Alveolar Septationmentioning

confidence: 99%

“…The following mouse (Mus musculus) strains were used: Rosa mTmG (Muzumdar et al, 2007), Rosa RG (Shioi et al, 2011), Rosa nTnG (Prigge et al, 2013), Rosa Sox2 (Lu et al, 2010), Rosa tdT (Madisen et al, 2010), Shh…”

Section: Micementioning

confidence: 99%

Development and plasticity of alveolar type 1 cells

et al. 2015

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Alveolar type 1 (AT1) cells cover >95% of the gas exchange surface and are extremely thin to facilitate passive gas diffusion. The development of these highly specialized cells and its coordination with the formation of the honeycomb-like alveolar structure are poorly understood. Using new marker-based stereology and single-cell imaging methods, we show that AT1 cells in the mouse lung form expansive thin cellular extensions via a non-proliferative two-step process while retaining cellular plasticity. In the flattening step, AT1 cells undergo molecular specification and remodel cell junctions while remaining connected to their epithelial neighbors. In the folding step, AT1 cells increase in size by more than 10-fold and undergo cellular morphogenesis that matches capillary and secondary septa formation, resulting in a single AT1 cell spanning multiple alveoli. Furthermore, AT1 cells are an unexpected source of VEGFA and their normal development is required for alveolar angiogenesis. Notably, a majority of AT1 cells proliferate upon ectopic SOX2 expression and undergo stage-dependent cell fate reprogramming. These results provide evidence that AT1 cells have both structural and signaling roles in alveolar maturation and can exit their terminally differentiated non-proliferative state. Our findings suggest that AT1 cells might be a new target in the pathogenesis and treatment of lung diseases associated with premature birth.

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Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei

Cited by 70 publications

References 43 publications

Genetic rescue models refute nonautonomous rod cell death in retinitis pigmentosa

Genetic rescue models refute nonautonomous rod cell death in retinitis pigmentosa

Hydrodynamic Delivery of Cre Protein to Lineage-Mark or Time-Stamp Mouse Hepatocytes In situ

Development and plasticity of alveolar type 1 cells

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