The tamoxifen-responsive conditional Cdh5-CreERT2 is commonly used for endothelial cell specific conditional deletion of loxP-flanked gene sequences. To address the role of endothelial cell Shb gene for B16F10 melanoma immune responses, tamoxifen-injected Cdh5-CreERT2/WT and Cdh5-CreERT2/Shbflox/flox mice received subcutaneous tumor cell injections. We observed a decrease of tumor myeloid cell Shb mRNA in the tamoxifen treated Cdh5-CreERT2/Shbflox/flox mice, which was not present when the mice had undergone a preceding bone marrow transplantation using wild type bone marrow. Differences in CD4+/FoxP3+ Tregs were similarly abolished by a preceding bone marrow transplantation. In ROSA26-mTmG mice, Cdh5-CreERT2 caused detectable floxing in certain bone marrow populations and in spleen cells. Floxing in bone marrow could be detected two months after tamoxifen treatment. In the spleen, however, floxing was undetectable two months after tamoxifen treatment, suggesting that Cdh5-CreERT2 is operating in a non-renewable population of hematopoietic cells in this organ. These data suggest that conditional gene deletion in hematopoietic cells is a potential confounder in experiments attempting to assess the role of endothelial specific effects. A cautious approach to achieve an endothelial-specific phenotype would be to adopt a strategy that includes a preceding bone marrow transplantation.

Cyrus Tang Hematology Center Soochow University Suzhou China

Department of Immunology Genetics and Pathology Uppsala University Uppsala Sweden

Department of Medical Cell Biology Uppsala University Uppsala Sweden

Institute of Molecular Genetics of the CAS Prague Czech Republic

Transgenic Animals Facility Oulu University Oulu Finland

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Welsh M, Jamalpour M, Zang G, Akerblom B. The role of the Src Homology-2 domain containing protein B (SHB) in beta cells. J Mol Endocrinol. 2016;56:R21–31. doi: 10.1530/JME-15-0228. PubMed DOI

Funa NS, et al. Dysfunctional microvasculature as a consequence of shb gene inactivation causes impaired tumor growth. Cancer research. 2009;69:2141–2148. doi: 10.1158/0008-5472.CAN-08-3797. PubMed DOI

Akerblom B, et al. Heterogeneity among RIP-Tag2 insulinomas allows vascular endothelial growth factor-A independent tumor expansion as revealed by studies in Shb mutant mice: implications for tumor angiogenesis. Molecular oncology. 2012;6:333–346. doi: 10.1016/j.molonc.2012.01.006. PubMed DOI PMC

Zang G, et al. Vascular dysfunction and increased metastasis of B16F10 melanomas in Shb deficient mice as compared with their wild type counterparts. BMC Cancer. 2015;15:234. doi: 10.1186/s12885-015-1269-y. PubMed DOI PMC

Li X, et al. Pro-tumoral immune cell alterations in wild type and Shb-deficient mice in response to 4T1 breast carcinomas. Oncotarget. 2018;9:18720–18733. doi: 10.18632/oncotarget.24643. PubMed DOI PMC

Gustafsson K, Jamalpour M, Trinh C, Kharas MG, Welsh M. The Src homology-2 protein Shb modulates focal adhesion kinase signaling in a BCR-ABL myeloproliferative disorder causing accelerated progression of disease. Journal of hematology & oncology. 2014;7:45. doi: 10.1186/1756-8722-7-45. PubMed DOI PMC

Jamalpour Maria, Li Xiujuan, Cavelier Lucia, Gustafsson Karin, Mostoslavsky Gustavo, Höglund Martin, Welsh Michael. Tumor SHB gene expression affects disease characteristics in human acute myeloid leukemia. Tumor Biology. 2017;39(10):101042831772064. doi: 10.1177/1010428317720643. PubMed DOI

Jamalpour Maria, Li Xiujuan, Gustafsson Karin, Tyner Jeffrey W, Welsh Michael. Disparate effects of Shb gene deficiency on disease characteristics in murine models of myeloid, B-cell, and T-cell leukemia. Tumor Biology. 2018;40(4):101042831877147. doi: 10.1177/1010428318771472. PubMed DOI

Christoffersson G, et al. Vascular adaptation to a dysfunctional endothelium as a consequence of Shb deficiency. Angiogenesis. 2012;15:469–480. doi: 10.1007/s10456-012-9275-z. PubMed DOI PMC

Nikpour M, et al. Shb deficiency in endothelium but not in leucocytes is responsible for impaired vascular performance during hindlimb ischaemia. Acta physiologica. 2015;214:200–209. doi: 10.1111/apha.12448. PubMed DOI

Anneren C, Lindholm CK, Kriz V, Welsh M. The FRK/RAK-SHB signaling cascade: a versatile signal-transduction pathway that regulates cell survival, differentiation and proliferation. Current molecular medicine. 2003;3:313–324. doi: 10.2174/1566524033479744. PubMed DOI

Gustafsson K, et al. Shb deficient mice display an augmented TH2 response in peripheral CD4+ T cells. BMC immunology. 2011;12:3. doi: 10.1186/1471-2172-12-3. PubMed DOI PMC

Gustafsson K, et al. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity. Experimental cell research. 2013;319:1852–1864. doi: 10.1016/j.yexcr.2013.03.020. PubMed DOI

Gustafsson K, Willebrand E, Welsh M. Absence of the adaptor protein Shb potentiates the T helper type 2 response in a mouse model of atopic dermatitis. Immunology. 2014;143:33–41. doi: 10.1111/imm.12286. PubMed DOI PMC

Zang G, et al. Aberrant association between vascular endothelial growth factor receptor-2 and VE-cadherin in response to vascular endothelial growth factor-a in Shb-deficient lung endothelial cells. Cellular signalling. 2013;25:85–92. doi: 10.1016/j.cellsig.2012.09.018. PubMed DOI

Sorensen I, Adams RH, Gossler A. DLL1-mediated Notch activation regulates endothelial identity in mouse fetal arteries. Blood. 2009;113:5680–5688. doi: 10.1182/blood-2008-08-174508. PubMed DOI

Tang Y, Harrington A, Yang X, Friesel RE, Liaw L. The contribution of the Tie2+ lineage to primitive and definitive hematopoietic cells. Genesis. 2010;48:563–567. doi: 10.1002/dvg.20654. PubMed DOI PMC

Chen MJ, Yokomizo T, Zeigler BM, Dzierzak E, Speck NA. Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter. Nature. 2009;457:887–891. doi: 10.1038/nature07619. PubMed DOI PMC

Monvoisin A, et al. VE-cadherin-CreERT2 transgenic mouse: a model for inducible recombination in the endothelium. Developmental dynamics: an official publication of the American Association of Anatomists. 2006;235:3413–3422. doi: 10.1002/dvdy.20982. PubMed DOI

Okabe K, et al. Neurons limit angiogenesis by titrating VEGF in retina. Cell. 2014;159:584–596. doi: 10.1016/j.cell.2014.09.025. PubMed DOI

Giladi A, et al. Single-cell characterization of haematopoietic progenitors and their trajectories in homeostasis and perturbed haematopoiesis. Nat Cell Biol. 2018;20:836–846. doi: 10.1038/s41556-018-0121-4. PubMed DOI

Kowalczyk MS, et al. Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells. Genome Res. 2015;25:1860–1872. doi: 10.1101/gr.192237.115. PubMed DOI PMC

Olsson A, et al. Single-cell analysis of mixed-lineage states leading to a binary cell fate choice. Nature. 2016;537:698–702. doi: 10.1038/nature19348. PubMed DOI PMC

Ulvmar MH, Martinez-Corral I, Stanczuk L, Makinen T. Pdgfrb-Cre targets lymphatic endothelial cells of both venous and non-venous origins. Genesis. 2016;54:350–358. doi: 10.1002/dvg.22939. PubMed DOI PMC

Kusumbe AP, et al. Age-dependent modulation of vascular niches for haematopoietic stem cells. Nature. 2016;532:380–384. doi: 10.1038/nature17638. PubMed DOI PMC

Kunisaki Y, et al. Arteriolar niches maintain haematopoietic stem cell quiescence. Nature. 2013;502:637–643. doi: 10.1038/nature12612. PubMed DOI PMC

Vanlandewijck M, et al. A molecular atlas of cell types and zonation in the brain vasculature. Nature. 2018;554:475–480. doi: 10.1038/nature25739. PubMed DOI

Vooijs M, Jonkers J, Berns A. A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent. EMBO Rep. 2001;2:292–297. doi: 10.1093/embo-reports/kve064. PubMed DOI PMC

Liu J, et al. Non-parallel recombination limits Cre-LoxP-based reporters as precise indicators of conditional genetic manipulation. Genesis. 2013;51:436–442. doi: 10.1002/dvg.22384. PubMed DOI PMC

Pietras EM, et al. Functionally Distinct Subsets of Lineage-Biased Multipotent Progenitors Control Blood Production in Normal and Regenerative Conditions. Cell Stem Cell. 2015;17:35–46. doi: 10.1016/j.stem.2015.05.003. PubMed DOI PMC

Hooper AT, et al. Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells. Cell Stem Cell. 2009;4:263–274. doi: 10.1016/j.stem.2009.01.006. PubMed DOI PMC

Skarnes WC, et al. A conditional knockout resource for the genome-wide study of mouse gene function. Nature. 2011;474:337–342. doi: 10.1038/nature10163. PubMed DOI PMC

Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. Genesis. 2007;45:593–605. doi: 10.1002/dvg.20335. PubMed DOI