Generation Of Vegfr3-Cre^ERT2 Transgenic Mice By Gene Knockin Strategy And Its Application

In mammals,lymphatic vessels mainly originate from veins.At the early embryonic stage,a proportion of venous endothelial cells commit to lymphatic endothelial cells and form the first lymphatic structures,lymph sacs.Following this,a mature lymphatic vessel network is generated by the process of lymphangiogenesis.During the past years,with the availability of new transgenic mouse models and better imaging tools,both physiological and pathological functions of the lymphatic system have been gradually revealed.It has been shown that lymphatic vessels are crucial for the regulation of fluid homeostasis,blood pressure,lipid metabolism,immunity,inflammation,and tumor metastasis.Based on the Cre/LoxP system,gene editing technology provides an effective tool for studying the biological function of genes in a temporally and spatially controlled manner.However,the achievement of efficient gene deletion depends on the expression level and tissue specificity of Cre transgenic mice.The major aim of this study was to establish a transgenic line overexpressing an inducible recombinase(CreERT2)for lymphatic research.Vascular Endothelial Growth Factor Receptor-3(VEGFR-3)is first expressed in blood vascular endothelial cells,but becomes restricted to lymphatic endothelial cells after midgestation during embryogenesis.To generate a transgenic line with the expression of Cre recombinase in lymphatic endothelial cells(Vegfr3-CreERT2),we inserted an IRES element(internal ribosomal entry site)together with the CreERT2 coding sequence after the stop codon in exon 30 of Vegfr3 gene.The CreERT2 fusion protein is composed of Cre recombinase and the mutated ligand binding domain of human estrogen receptor.We then crossed the Vegfr3-CreERT2 mice with Rosa26-mTmG reporter mice to generate the doubly transgenic line(Vegfr3-CreERT2;Rosa26-mTmG).The expression of enhanced green fluorescent protein(eGFP),resulting from the CreERT2 mediated excisioU of tdTomato(mT)cassette,was induced by the treatment with tamoxifen(from E13.5-15.5).The expression and distribution of CreERT2 recombinase was reflected by eGFP signals in the Vegfr3-CreERT2 transgenic mice.We found that during embryonic development,eGFP+lymphatic endothelial cells was detected in all the tissues examined including abdominal skin,mesentery and heart of Vegfr3-CreERT2;Rosa26-mTmG mice,particularly with strong expression in valves of collecting lymphatic vessels.In addition,we performed the whole-mount immunofluorescence analysis of eGFP signals to monitor lymphatic development in lungs at different stages of embryogenesis(including E12.5,E13.5,E14.5,E16.5 and E18.5).Unfortunately,due to the weak eGFP signals in the deep layer of lung tissues,we only managed to image the superficial lymphatic network in left lung of Vegfr3-CreERT2;Rosa26-mTmG mice.At the postnatal stage,tamoxifen treatment(postnatal day 1 to 4 days,named P1-P4)also effectively induced the expression of eGFP in lymphatic endothelial cells in the abdominal and tail skin,lung and trachea of neonatal mice(P7).Consistent with the above results,we used tamoxifen to treat mice at three-week-old and adult stages,eGFP signals could be detected in lymphatic vessels of all the tissues examined,such as the ear ventral skin,trachea and diaphragm,and especially in valves of collecting lymphatic vessels.Furthermore,we found that at both embryonic and postnatal stages,eGFP expression was induced by the tamoxifen treatment in some blood vascular endothelial cells of Vegfr3-CreERT2;Rosa26-mTmG mice.There was widespread expression of eGFP in liver sinusoid vessels,which also reflected their expression of VEGFR-3.eGFP positive blood vascular endothelial cells were also detected in villi and retina.Interestingly,a few eGFP+ cells were found in large vessels including veins and arteries of retina.The biological significance of VEGFR3 expression in blood vessels requires further investigation.In summary,we have successfully generated a transgenic Cre mouse line(Vegfr3-CreERT2)by the knockin strategy.Treatment with tamoxifen effectively induced the reporter gene(eGFP)expression in lymphatic endothelial cells of all the tissues examined at different developmental stages.Furthermore,consistent with the expression of endogenous VEGFR-3,eGFP signals were also detected in some blood vascular endothelial cells.Therefore,the Vegfr3-CreERT2 transgenic line could be a useful tool to analyze the heterogeneity of blood vascular endothelial cells in tissues.