Real-time monitoring of keratin 5 expression during burn re-epithelialization1

Bruen, Kevin J; Campbell, Chris A.; Schooler, Wesley; deSerres, Suzan; Cairns, Bruce A.; Hultman, C. Scott; Meyer, Anthony A.; Randell, Scott H.

doi:10.1016/j.jss.2004.02.024

Cited by 19 publications

(14 citation statements)

References 24 publications

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“…Both the bovine K5 promoter and the human K18 sequence used in this study have been used successfully by others to target expression of other transgenes, including EGFP, appropriately to K5- and K18-expressing cells in mice (Bruen et al 2004; Liang et al 2009; Wen et al 2003). We chose to generate transgenic models using K5 and K18 regulatory sequences to control CFP and RFP because these reporter proteins are spectrally distinct, thus allowing for their combined functionality in double transgenic mice.…”

Section: Discussionmentioning

confidence: 99%

Epithelial cell-targeted transgene expression enables isolation of cyan fluorescent protein (CFP)-expressing prostate stem/progenitor cells

2011

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To establish a method for efficient and relatively easy isolation of a cell population containing epithelial prostate stem cells, we developed two transgenic mouse models, K5/CFP and K18/RFP. In these models, promoters of the cytokeratin 5 (Krt5) and the cytokeratin 18 (Krt18) genes regulate cyan and red fluorescent proteins (CFP and RFP), respectively. CFP and RFP reporter protein fluorescence allows for visualization of K5+ and K18+ epithelial cells within the cellular spatial context of the prostate gland and for their direct isolation by FACS. Using these models, it is possible to test directly the stem cell properties of prostate epithelial cell populations that are positively selected based on expression of cytoplasmic proteins, K5 and K18. After validating appropriate expression of the K5/CFP and K18/RFP transgenes in the developing and adult prostate, we demonstrate that a subset of CFP-expressing prostate cells exhibits stem cell proliferation potential and differentiation capabilities. Then, using prostate cells sorted from double transgenic mice (K5/CFP + K18/RFP), we compare RNA microarrays of sorted K5+K18+ basal and K5−K18+ luminal epithelial cells, and identify genes that are differentially expressed. Several genes that are over-expressed in K5+ cells have previously been identified as potential stem cell markers. These results suggest that FACS isolation of prostate cells from these mice based on combining reporter gene fluorescence with expression of potential stem cell surface marker proteins will yield populations of cells enriched for stem cells to a degree that has not been attained by using cell surface markers alone.

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Section: Discussionmentioning

confidence: 99%

Epithelial cell-targeted transgene expression enables isolation of cyan fluorescent protein (CFP)-expressing prostate stem/progenitor cells

2011

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“…Other reporter mouse models, exhibiting GFP expression in a restricted and interesting pattern within skin epithelia, have been devised (5,35,45,71). In two instances, fluorescenceactivated cell sorting was exploited to isolate and characterize populations of hair follicle cells enriched in epithelial stem cells (69,71).…”

Section: Discussionmentioning

confidence: 99%

Exploiting the Keratin 17 Gene Promoter To Visualize Live Cells in Epithelial Appendages of Mice

Bianchi

DePianto²,

McGowan³

et al. 2005

Molecular and Cellular Biology

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Keratin genes afford, given their large number (>50) and differential regulation, a unique opportunity to study the mechanisms underlying specification and differentiation in epithelia of higher metazoans. Moreover, the small size and regulation in cis of many keratin genes enable the use of their regulatory sequence to achieve targeted gene expression in mice. Here we show that 2 kilobases of 5 upstream region from the mouse keratin 17 gene (mK17) confers expression of green fluorescent protein (GFP) in major epithelial appendages of transgenic mice. Like that of mK17, onset of [mK17 5]-GFP reporter expression coincides with the appearance of ectoderm-derived epithelial appendages during embryonic development. In adult mice, [mK17 5]-GFP is appropriately regulated within hair, nail, glands, and oral papilla. Tracking of GFP fluorescence allows for the visualization of growth cycle-related changes in hair follicles, and the defects engendered by the hairless mutation, in live skin tissue. Deletion of an internal 48-bp interval, which encompasses a Gli-responsive element, from this promoter results in loss of GFP fluorescence in most appendages in vivo, suggesting that sonic hedgehog participates in K17 regulation. The compact mK17 gene promoter provides a novel tool for appendage-preferred gene expression and manipulation in transgenic mice.

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“…Control mice were outbred ICR. The Krt5-eGFP transgenic mouse line, in which a 6-kb fragment of the human basal cell-specific KRT5 promoter drives the expression of EGFP, has been described (Bruen et al 2004;Schoch et al 2004). This line was originally derived on the (C57Bl/6xC3H) hybrid background and is maintained by interbreeding.…”

Section: Methodsmentioning

confidence: 99%

Culture of endodermal stem/progenitor cells of the mouse tongue

Luo

Okubo

Randell

et al. 2008

In Vitro Cell.Dev.Biol.-Animal

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The tongue represents a very accessible source of tissue-specific epithelial stem cells of endodermal origin. However, little is known about the properties of these cells and the mechanisms regulating their proliferation and differentiation. Foxa2, an endodermal marker, is expressed throughout the tongue epithelium during embryonic development but becomes confined to a minority of basal cells and some taste bud sensory cells in the adult tongue. Using a previously described line of transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed under the control of a human keratin 5 promoter region (Krt5-eGFP), we have isolated a subpopulation of cells in the basal epithelial layer of the mouse tongue with a high efficiency of generating holoclones of undifferentiated cells in culture with a feeder layer. Krt5-GFP(hi) cells can both self renew and give rise to differentiated stratified keratinized epithelial cells when cultured on an air-liquid interface.

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Real-time monitoring of keratin 5 expression during burn re-epithelialization1

Cited by 19 publications

References 24 publications

Epithelial cell-targeted transgene expression enables isolation of cyan fluorescent protein (CFP)-expressing prostate stem/progenitor cells

Epithelial cell-targeted transgene expression enables isolation of cyan fluorescent protein (CFP)-expressing prostate stem/progenitor cells

Exploiting the Keratin 17 Gene Promoter To Visualize Live Cells in Epithelial Appendages of Mice

Culture of endodermal stem/progenitor cells of the mouse tongue

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