Real-time monitoring of keratin 5 expression during burn re-epithelialization
ABSTRACT Keratin is a major protein produced during epithelialization following burn injury and is a useful marker for assessing wound healing. Transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the keratin 5 (K5) promoter (K5GFP mice) were used to monitor keratin expression, and thus, re-epithelialization of burn wounds.
K5GFP transgenic mice were created using conventional techniques, with PCR and Southern blot confirmation of transgene incorporation, followed by selection of the line with the most intense and consistent basal epithelial EGFP expression. Epi-fluorescent microscopy of 24 K5GFP mouse flanks and 10 negative littermate controls was used to characterize EGFP intensity, before wounding and serially for 30 days after administration of a standardized burn wound and excision. Biopsy sections of K5GFP and negative control mice were stained with K5 antibody and imaged with confocal microscopy to characterize the distribution of EGFP and K5 at baseline and after injury and to examine the correlation between K5 expression and EGFP expression during healing.
Green fluorescence intensity increased at the advancing wound margin of burned K5GFP mice, reaching a maximum between days 12 and 15 post-burn and then decreasing as healing completed. K5 and EGFP expression increased in parallel in burned K5GFP mice as demonstrated by confocal microscopy.
EGFP expression correlated with K5 expression during wound healing and therefore serves as a good marker of re-epithelialization. This transgenic model allows noninvasive, real-time assessment of in vivo K5 expression and will be useful in the study of wound healing.
- SourceAvailable from: Jiake Xu
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- "Full-thickness burn wounds were generated following a validated protocol as previously described   . Briefly, 9-week old wild type C57BL/6 female mice were anaesthetised in a closed chamber with a continuous flow of 4% isoflurane and high flow oxygen. "
ABSTRACT: Bone loss after severe burn injury is well established, and is thought to be a consequence of the severe hyper-metabolic response as well as changes in cytokine and glucocorticoid levels that decrease bone synthesis and increase rate of loss. However, 90% of presentations are for non-severe burns which do not elicit this response. Little is known about whether these non-severe injuries may also affect bone tissue, and whether other mechanisms may be involved. To investigate whether bone loss occurs after a non-severe burn injury we used a mouse model of an approximately 8% total body surface area (TBSA) full-thickness burn and micro-CT. We also assessed whether blocking TNF-α after a burn injury by administration of an antibody could modulate the impacts of the burn on bone tissue. There was a significant loss of trabecular bone volume of (3.27% compared to 5.27%, p = 0.0051) after non-severe burn injury. Trabecular number was significantly decreased (0.57/mm after injury compared to 1.02/mm controls, p = 0.0051) and spacing increased after burn injury (0.40 compared to 0.28, p = 0.0083). Anti-TNF-α antibodies significantly improved trabecular bone volume (8.53%, p = 0.0034) and number after burn injury (1.28/mm, p = 0.0034). There was no significant change observed in cortical bone after burn injury or administration of anti-TNF-α antibodies. These findings show that non-severe burn injury can lead to changes in bone metabolism. Monitoring bone density in patients with non-severe injuries and interventions to limit the impacts of the inflammatory storm may benefit patient recovery and outcomes.Burns 10/2014; 41(3). DOI:10.1016/j.burns.2014.09.004 · 1.84 Impact Factor
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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. "
ABSTRACT: 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.Transgenic Research 10/2011; 20(5):1073-86. DOI:10.1007/s11248-010-9478-2 · 2.28 Impact Factor
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ABSTRACT: 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.Molecular and Cellular Biology 09/2005; 25(16):7249-59. DOI:10.1128/MCB.25.16.7249-7259.2005 · 5.04 Impact Factor