Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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Development of a Novel Lymphatic Reporter Mouse
Gina Farias-Eisner, BA, Daniel A. Cuzzone, MD, Seth Z. Aschen, BS, Nicholas J. Albano, BS, Swapna Ghanta, MD, Evan Weitman, MD, Babak Mehrara, MD.
Memorial Sloan Kettering Cancer Setting, New York, NY, USA.

PURPOSE:
The lymphovascular system is a highly specialized organ system that is responsible for interstitial fluid homeostasis and immune function. The lymphatic endothelial cell, the basic subunit of the lymphovascular system, possesses highly conserved proteins that have been targets for identifying lymphatics. These include the hyaluronan receptor lymphatic vessel endothelial receptor 1 (LYVE-1), the membrane glycoprotein podoplanin, the transcription factor Prox-1 and the VEGF-R3 tyrosine kinase. Identification of the lymphatic vessels has been instrumental in studying various congenital disease states as well as malignancy. Conventional methods for identification can be harsh and caustic. Here we describe a novel reporter mouse that displays high fidelity for lymphovascular identification.
METHODS:
Transgenic lymphatic reporter mice using a tamoxifen-inducible Cre-Lox system were developed. This was achieved by crossing C57B6 transgenic mice that expressed the bacterial Cre recombinase gene driven by the mouse VEGF-R3 (FLT4) under the control of tamoxifen, with C57B6 LacZ-LoxP transgenic mice. To activate Cre-lox recombination, homozygous FLT4-Cre/LacZ-LoxP mice (FLT4-LacZ mice) were injected intraperitoneally with 200mg/kg tamoxifen for 5 days followed by sacrifice. LacZ expression was visualized by incubating tissues in Lac-Z buffer solution, followed by embedding and sectioning. To analyze the penetrance/specificity of Cre-expression, tissues were stained using antibodies directed against the LYVE-1 or Von-Willibrand factor (VWF), a marker of blood vessels.
RESULTS:
Histological analysis of skin sections revealed that the expression of Flt4-Cre was tamoxifen dependent. Control mice that were not primed with tamoxifen prior to sacrifice showed no B-gal staining in LYVE-1+ lymphatic vessels. In contrast, treatment of transgenic mice with tamoxifen resulted in high-level expression of Cre-recombinase with more than 90% of LYVE-1+ vessels demonstrating B-gal staining. The expression of B-gal was limited to lymphatic vessels because we found no B-gal staining of blood vessels stained with Von Willebrand factor.
CONCLUSION:
Here we describe the development of a novel reporter mouse for identifying lymphatic vessels. There is very high fidelity in the identification of lymphovascular structures. This reporter mouse has multiple applications for studies utilizing models of lymphatic manipulation.


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