Abstract
Lymphatic vessels are indispensable for tissue fluid homeostasis, transport of solutes and dietary lipids and immune cell trafficking. In contrast to blood vessels, which are easily visible by their erythrocyte cargo, lymphatic vessels are not readily detected in the tissue context. Their invisibility interferes with the analysis of the three-dimensional lymph vessel structure in large tissue volumes and hampers dynamic intravital studies on lymphatic function and pathofunction. An approach to overcome these limitations are mouse models, which express transgenic fluorescent proteins under the control of tissue-specific promotor elements. We introduce here the BAC-transgenic mouse reporter strain Vegfr3-tdTomato that expresses a membrane-tagged version of tdTomato under control of Flt4 regulatory elements. Vegfr3-tdTomato mice inherited the reporter in a mendelian fashion and showed selective and stable fluorescence in the lymphatic vessels of multiple organs tested, including lung, kidney, heart, diaphragm, intestine, mesentery, liver and dermis. In this model, tdTomato expression was sufficient for direct visualisation of lymphatic vessels by epifluorescence microscopy. Furthermore, lymph vessels were readily visualized using a number of microscopic modalities including confocal laser scanning, light sheet fluorescence and two-photon microscopy. Due to the early onset of VEGFR-3 expression in venous embryonic vessels and the short maturation time of tdTomato, this reporter offers an interesting alternative to Prox1-promoter driven lymphatic reporter mice for instance to study the developmental differentiation of venous to lymphatic endothelial cells.
Highlights
The lymphatic vessel system consists of a blind-ending network of capillaries that take up interstitial fluid, solutes, dietary lipids and cells, which together constitute the lymph
Lymph is routed via collecting lymphatic vessels to connections with
The resulting primary cell cultures were comprised of a mixture of endothelial cells (ECs) of blood and lymphatic origin
Summary
We have flanked a cDNA, coding for the membrane-tagged RFP-derivate tdTomato-CAAX followed by an ampicillin-resistance cassette for positive selection with homology regions of the murine Flt gene. Targeting vectors were sequenced to verify correct insertion of the DNA fragments. We used Red/ET recombineering in E. coli DH10B to generate a modified bacterial artificial chromosome (the BAC clone RP23-58E13, harbouring the Flt gene within 214kB of genomic sequence of mouse chromosome 11). The construct was designed such that the VEGFR-3 start codon became the tdTomato start codon. Transgenic founders were identified by genomic PCR analysis (PCR1: fwd 5’GACAACAACATGGCCGTCA-3’, rev 5’-CTTGTACAGCTCGTCCATGC-3’ and PCR2: fwd 5’- GCTCTCACTCCCAGCCTAG-3’, rev 5’-ACTCTTTGATGACGGCCATGTT-3’) and were subsequently screened for Vegfr promotor-driven tdTomato expression by stereomicroscopy of the dermal lymphatic vascular plexus in ear skin biopsies. One of three founders was selected for line establishment, based on the brightness of tdTomato fluorescence
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