Konference: 2011 7. Sympozium a workshop molekulární patologie a histo-cyto-chemie
Kategorie: Nádorová biologie/imunologie/genetika a buněčná terapie
Téma: Postery
Číslo abstraktu: 004p
Autoři: P. Kasparek; S. Suchanova; P. Křenek; R. Sedláček
Introduction: A number of skin diseases
and pathological conditions such as dermatitis, psoriasis, or skin
wounding are characterized by abnormal maturation and proliferation
of keratinocytes. Thus, monitoring of keratinocyte differentiation
in vivo appears to be a valuable tool to study processes of
epidermal physiology and pathology. To address this issue, we
generated a transgenic mouse model expressing a fluorescent marker
tdTomato under the control of modified human involucrin promoter.
Involucrin is well established differentiation marker being
expressed in suprabasal layers of epidermis and in
hyperproliferative keratinocytes of healing wounds.
Aim: To create a model to study patho-physiological processes in vitro and in vivo in the outmost layers of epidermis.
Materials and methods: DNA construct containing truncated promoter and first intron of human involucrin, cDNA of tdTomato, and polyA signal was subcloned and used for generation of transgenic mice by pronuclear microinjection on C57Bl/6N background. Skin from mouse ears and paws was cryosectioned and the expression of the transgene compared to the expression of other differentiation and proliferation markers using involucrin, keratin 14, keratin 6 antibodies. Model of irritant dermatitis was induced by application of croton oil. Skin wound was induced on mouse ear by linear 3 mm-puncher. To study regulation of involucrin promoter in vivo, transgenic mice were depilated and analyzed by whole-body imaging.
Results: We have generated a transgenic mouse expressing tdTomato, a fluorescent reporter, under the control of human involucrin promoter. To assess the specific targeting and regulation of transgene, multiple organs were isolated from positive animals and analyzed for tdTomato signals using fluorescent microscopy. The truncated human involucrin promoter with its intron targeted the transgene efficiently and specifically into the uppermost epidermal layers as well as into epithelia of the tongue and bladder. Detail analyses of skin sections from transgenic animals revealed specific expression of tdTomato in differentiated keratinocytes. Induction of irritant dermatitis on mouse ear led to an increase of fluorescent signal in 24 hours. In the model of wound healing the fluorescent reporter was clearly upregulated 24 hours post wounding and this augmented expression lasted for additional 96 hours with the peak of expression around 72 hours. These observations are in accordance with previously reported changes of epidermal differentiation during acute dermatitis and healing of skin wounds. Fluorescence signal of the reporter can be easily followed in vivo using whole-body imaging.
Conclusions: The transgenic mouse expressing tdTomato reporter under the human involucrin promoter could be used as a valuable tool to study processes leading to the dysregulation of epidermal barrier. Keratinocytes undergoing rapid proliferation and differentiation are in transgenic animals characterized by increase of fluorescent signal, which can be easily detected in vivo. This novel mouse model can be further used to follow an effect of various therapeutic agents for treatment of skin diseases in vivo.
Aim: To create a model to study patho-physiological processes in vitro and in vivo in the outmost layers of epidermis.
Materials and methods: DNA construct containing truncated promoter and first intron of human involucrin, cDNA of tdTomato, and polyA signal was subcloned and used for generation of transgenic mice by pronuclear microinjection on C57Bl/6N background. Skin from mouse ears and paws was cryosectioned and the expression of the transgene compared to the expression of other differentiation and proliferation markers using involucrin, keratin 14, keratin 6 antibodies. Model of irritant dermatitis was induced by application of croton oil. Skin wound was induced on mouse ear by linear 3 mm-puncher. To study regulation of involucrin promoter in vivo, transgenic mice were depilated and analyzed by whole-body imaging.
Results: We have generated a transgenic mouse expressing tdTomato, a fluorescent reporter, under the control of human involucrin promoter. To assess the specific targeting and regulation of transgene, multiple organs were isolated from positive animals and analyzed for tdTomato signals using fluorescent microscopy. The truncated human involucrin promoter with its intron targeted the transgene efficiently and specifically into the uppermost epidermal layers as well as into epithelia of the tongue and bladder. Detail analyses of skin sections from transgenic animals revealed specific expression of tdTomato in differentiated keratinocytes. Induction of irritant dermatitis on mouse ear led to an increase of fluorescent signal in 24 hours. In the model of wound healing the fluorescent reporter was clearly upregulated 24 hours post wounding and this augmented expression lasted for additional 96 hours with the peak of expression around 72 hours. These observations are in accordance with previously reported changes of epidermal differentiation during acute dermatitis and healing of skin wounds. Fluorescence signal of the reporter can be easily followed in vivo using whole-body imaging.
Conclusions: The transgenic mouse expressing tdTomato reporter under the human involucrin promoter could be used as a valuable tool to study processes leading to the dysregulation of epidermal barrier. Keratinocytes undergoing rapid proliferation and differentiation are in transgenic animals characterized by increase of fluorescent signal, which can be easily detected in vivo. This novel mouse model can be further used to follow an effect of various therapeutic agents for treatment of skin diseases in vivo.
Datum přednesení příspěvku: 29. 4. 2011
