Sunday, October 28, 2007
13480

The Generation and Characterization of Transgenic Mice Overexpressing CD109 in the Skin

Joshua Vorstenbosch, BSc, Genevieve Chaput, MD, Stephane Roy, PhD, Lucie Lessard, MD, FRSC, and Anie Philip, PhD.

Introduction/Purpose: Few effective treatment strategies exist for wound healing pathologies such as hypertrophic scarring, keloids, and chronic wounds despite the fact that these disorders affect approximately 15% of the population. A possible solution to this problem involves altering the expression of cytokines such as TGF-beta at the wound site in an attempt to make adult wounds biochemically mimic embryonic, scarless wound healing. TGF- beta1 is a profibrotic growth factor that plays an important role throughout the entire wound healing response and its excessive action is thought to account for the abnormal scarring associated with skin pathologies. The correlation between excessive scarring and TGF- beta1 activity suggests that increased expression of a TGF- beta1 antagonist in the wound could diminish scar formation. Our lab has identified CD109 as a novel TGF- beta1 accessory receptor and shown that it functions to inhibit TGF- beta1 signaling. These data support the hypothesis that overexpression of CD109 in the skin will antagonize the effects of TGF- beta1, thereby reducing the extent of scarring and offering promise for a novel treatment strategy for wound healing pathologies.

Methods: To generate transgenic mice overexpressing CD109 in the skin, we created a DNA construct using Gateway technology from Invitrogen. We cloned the CD109 gene downstream of the K14 promoter in order to direct expression of CD109 specifically in the skin. The DNA construct was microinjected into FVB embryonic cells at the McGill Transgenic Core Facility and the cells were implanted into the uteri of pseudopregnant female FVB mice. Founder mice were confirmed by Southern Blot and subsequently bred to produce more transgenic mice. The genomic DNA of the F1 progeny was analyzed by PCR to confirm presence of the transgene with select mice later being sacrificed to screen for expression by Western Blot.

Results: Preliminary results demonstrate that the transgenic mice show an increase of expression of CD109 in the skin compared to wild-type controls and also exhibit more CD109 expression in the skin than in their brain, liver, and kidney.

Conclusions: The transgenic mice generated show overexpression of CD109 spatially restricted to the skin meeting the requirements of an in vivo model. These mice provide an ideal model to delineate the role of CD109, a TGF-beta antagonist, in reducing scarring. Specifically, this model will allow biochemical and morphological quantification of scarring parameters including collagen deposition, angiogenesis, reepithelialization and extracellular matrix remodeling to provide insight into the role of CD109 in wound healing and its efficacy as a novel treatment for wound healing pathologies.


View Synopsis (.doc format, 300.0 kb)