Development of a new method for isolation and long-term culture of organ-specific blood vascular and lymphatic endothelial cells of the mouse Takashi Yamaguchi, Taeko Ichise, Osamu Iwata, Akiko Hori, Tomomi Adachi, Masaru Nakamura, Nobuaki Yoshida and Hirotake Ichise Laboratory of Gene Expression and Regulation, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Japan Keywords Cre ⁄ loxP recombination; endothelial cell culture; endothelial heterogeneity; SV40 tsA58 large T antigen; transgenic mouse Correspondence H Ichise, Laboratory of Gene Expression and Regulation, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Fax: +81 5449 5455 Tel: +81 5449 5754 E-mail: h-ichise@ims.u-tokyo.ac.jp (Received 27 November 2007, revised 13 February 2008, accepted 22 February 2008) doi:10.1111/j.1742-4658.2008.06353.x Endothelial cells are indispensable components of the vascular system, and play pivotal roles during development and in health and disease Their properties have been studied extensively by in vivo analysis of genetically modified mice However, further analysis of the molecular and cellular phenotypes of endothelial cells and their heterogeneity at various developmental stages, in vascular beds and in various organs has often been hampered by difficulties in culturing mouse endothelial cells In order to overcome these difficulties, we developed a new transgenic mouse line expressing the SV40 tsA58 large T antigen (tsA58T Ag) under the control of a binary expression system based on Cre ⁄ loxP recombination tsA58T Ag-positive endothelial cells in primary cultures of a variety of organs proliferate continuously at 33 °C without undergoing cell senescence The resulting cell population consists of blood vascular and lymphatic endothelial cells, which could be separated by immunosorting Even when cultured for two months, the cells maintained endothelial cell properties, as assessed by expression of endothelium-specific markers and intracellular signaling through the vascular endothelial growth factor receptors VEGFR–2 and VEGFR-3, as well as their physiological characteristics In addition, lymphatic vessel endothelial hyaluronan receptor-1 (Lyve-1) expression in liver sinusoidal endothelial cells in vivo was retained in vitro, suggesting that an organ-specific endothelial characteristic was maintained These results show that our transgenic cell culture system is useful for culturing murine endothelial cells, and will provide an accessible method and applications for studying endothelial cell biology As an indispensable component of the vascular system, endothelial cells (ECs) have pivotal roles in development and in health and disease [1] Their properties have been studied by a combination of in vitro analyses of human primary ECs and in vivo analyses of genetically modified mice exhibiting vascular phenotypes Human primary ECs are well-established resources and are suitable for studying signal transduction and cellular physiology in vitro However, it is still difficult to control their gene expression strictly by current overexpression and knockdown procedures In addition, they are not representative of all types of ECs at various developmental stages and in vascular beds [2] On the other hand, the use of genetically Abbreviations BEC, blood vascular endothelial cell; DiI, 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate; EC, endothelial cell; ESC, embryonic stem cell; HRP, horseradish peroxidase; LDL, low-density lipoprotein; LEC, lymphatic endothelial cell; Lyve-1, lymphatic vessel endothelial hyaluronan receptor-1; MACS, magnetic-activated cell separation; MAPK, mitogen-activated protein kinase; PFA, paraformaldehyde; Prox-1, prospero-related homeobox-1; SV40T Ag, SV40 large T antigen; tsA58T Ag, large T antigen of SV40 mutant strain tsA58; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor 1988 FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS T Yamaguchi et al modified mice has accelerated the understanding of genetic mechanisms of endothelial development and functions However, further analyses of vascular phenotypes in vivo have been hampered by the complicated relationship between ECs and non-ECs such as mural, hematopoietic and mesenchymal fibroblast cells, even though a conditional genetic modification such as endothelium-specific knockouts can provide a partial solution to this problem Therefore, the isolation and maintenance of murine endothelial cells from various developmental stages and locations is important for dissecting molecular and cellular mechanisms of endothelial development and function Murine primary cells, including ECs, have a more limited growth potential than human primary cells Thus, ‘immortalization’ techniques have been strongly recommended for most analyses that require a large quantity of transcripts, proteins or cells For immortalization of ECs, viral oncogenic proteins have been used in previous studies The polyoma middle T antigen (PyMT Ag) allows selective proliferation of ECs in mixed-cell populations [3–5], aiding in analyses of genetically modified ECs in vitro [6–11] However, PyMT Ag causes endothelioma or hemangioma in vivo [3] and mimics activated receptor tyrosine kinases [12], which might obscure the analysis of endogenous receptor-mediated signaling Alternatively, tsA58T Ag, a mutated SV40T Ag leading to temperature-dependent, cell-type-independent cell proliferation [13,14], has been used for ‘conditional immortalization’ of ECs of wild-type and genetically modified mice [15–22] Despite the fact that tsA58T Ag-directed immortalization of ECs has been demonstrated, the method has been under-utilized due to the specialized techniques and expertise that are required for immunological isolation of ECs [23,24] to prevent proliferation of tsA58T Ag-expressing non-ECs Results and Discussion Generation of a transgenic mouse line carrying the CAG-bgeo-tsA58T Ag transgene In order to circumvent the problems described above, we developed a new transgenic mouse line expressing tsA58T Ag under the control of a binary expression system based on Cre ⁄ loxP recombination To obtain a transgenic mouse line with the potential to express tsA58T Ag in a variety of tissues including ECs, we exploited embryonic stem cell (ESC)-mediated transgenesis Briefly, we constructed a transgene driven by the CAG promoter [25] that expresses the b–geo gene [26] in the absence of Cre recombinase, but expresses A new method for mouse endothelial cell culture CAG loxP βgeo pA tsA58T loxP CAG pA tsA58T T26 Tg T26/Tie2-Cre Tg Tie2-Cre Tg Enzymatic digestion of organs Culture at 33 °C day tsA58T Ag-expressing endothelial cell Serial passages every 2–3 days at split ratio : day 20–30 Fig An endothelial cell culture scheme based on endotheliumspecific expression of tsA58T antigen pA, polyadenylation signal sequence; Tg, transgenic mouse the tsA58T Ag gene after Cre-mediated excision of the lox P-flanked b–geo gene (Fig 1) The plasmid vector-free transgene was introduced into ESCs, and G418-resistant clones were selected We next performed 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside (X-gal) staining of embryoid bodies derived from each clone and screened for the expression pattern of b–geo in the embryoid bodies Clone T26 had the most favorable b–geo expression pattern among the G418resistant clones (data not shown) tsA58T Ag expression in ESCs after Cre-mediated excision was verified by Western blotting (data not shown) The T26 transgenic mouse line was obtained through germline transmission from chimeric mice They grew normally, were fertile, and did not display any defects Endothelium-specific expression of tsA58T Ag in the transgenic mouse We next crossed female T26 transgenic mice with male Tie2–Cre transgenic mice [27], which removed a loxP-flanked DNA fragment in endothelial cells and FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS 1989 A new method for mouse endothelial cell culture T Yamaguchi et al hematopoietic cells (Fig 1) The resulting T26 ⁄ Tie2– Cre double-transgenic mice were born and grew normally, but died suddenly within 6–12 weeks after birth To determine whether the expression of tsA58T Ag was induced in ECs, we performed immunohistoSV40T CD31 B SV40T CD31 SV40T Lyve-1 Liver Heart Yolk sac Brain Embryo A chemistry using antibodies against the pan-EC marker, CD31, the lymphatic endothelial and liver sinusoidal endothelial marker Lyve-1 (lymphatic vessel endothelial hyaluronan receptor-1) [28–32] and SV40T Ag Immunostaining revealed that tsA58T Ag was Thymus Lung Uterus SV40T CD31 Cardiac valve C Fig Expression pattern of tsA58T Ag in T26 ⁄ Tie2–Cre double-transgenic mice (A) tsA58T Ag (red) was expressed in CD31-positive ECs (green) of an E9.5 T26 ⁄ Tie2–Cre double-transgenic embryo and its yolk sac (B) tsA58T Ag (red) was expressed in CD31-positive ECs (green, left panels) and Lyve-1-positive ECs (green, right panels) of 3)6-week-old T26 ⁄ Tie2–Cre double-transgenic mice Lyve-1-positive ECs were not detected in the brain (top right), which is known to be an LEC-free organ (C) tsA58T Ag (red) was also expressed in non-endothelial cells of the thymic medulla and interstitial cells of the cardiac valve Arrowheads indicate CD31-positive ECs (green) All micrographs are shown at the same magnification Scale bar = 50 lm 1990 FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS T Yamaguchi et al expressed in CD31-positive ECs of E9.5 embryos proper and yolk sacs (Fig 2A) Postnatally, tsA58T Ag was not only expressed in CD31-positive ECs in the brain, heart, lung, liver and uterus (Fig 2B), but was also expressed in Lyve-1-positive lymphatic endothelial cells (LECs) in the heart, lung and uterus, and sinusoidal ECs in the liver of 3–6-week-old doubletransgenic mice (Fig 2B), indicating that endothelium-specific expression of tsA58T Ag was achieved as expected Despite the mortality of the young double-transgenic mice, no gross abnormalities, such as endothelial hyperplasia, dysplasia or bleeding, could be found in live or dead double-transgenic mice However, immunostaining revealed that tsA58T Ag was expressed in non-ECs, including a subset of thymocytes and cardiac valvular cells (Fig 2C) These observations are comparable to those of previous studies using the same Tie2–Cre transgenic mouse line, which showed that recombination occurred in hematopoietic cells as well as ECs [27], and that cardiac valvular cells were derived from endothelial cells through an endothelial-to-mesenchymal transition during early development [33] The presence of these cells may cause a dysfunctional cardiac flow and cause the sudden death of the transgenic mice, although it remains to be determined whether T antigen-expressing cardiac valves are functionally affected A new method for mouse endothelial cell culture genic mice did not grow beyond 2–3 weeks (data not shown), confirming that tsA58T Ag-directed proliferation was only achieved by Cre-mediated excision Characterization of tsA58T Ag-expressing endothelial cell populations In order to examine whether the tsA58T Ag-positive cells maintained EC properties, we first performed immunocytochemistry for EC markers and assessed the uptake of acetylated low-density lipoproteins (LDLs) The cell populations derived from the brain, lung, heart, liver and uterus stained positive for CD31 (Fig 3B for the brain, liver and uterus; data not shown for the lung and heart), strongly suggesting that the tsA58T Ag-positive cells originated from ECs A subset of the cell populations from the lung and heart (data not shown) and a A DAPI tsA58T Ag Merge B Brain Liver Uterus C Brain Liver Uterus D Brain Uterus Brain Endothelial cell culture from organs of T26/Tie2–Cre double-transgenic mice Following the demonstration of endothelium-specific expression of tsA58T Ag in vivo, we performed primary cell culturing (Fig 1) Several organs (the brain, heart, lung, liver and uterus) were obtained from 3-week-old T26 single- or T26 ⁄ Tie2–Cre double-transgenic mice, dissected, and dissociated by enzymatic digestion Dispersed cell suspensions were plated onto gelatin-coated plastic dishes and cultured at 33 °C (day in Fig 1) For the initial weeks, primary cells, including both tsA58T Ag-negative cells (primarily fibroblasts) and tsA58T Ag-positive cells, proliferated, and ECs could barely be morphologically distinguished However, tsA58T Ag-negative cells gradually stopped proliferating and underwent senescence at about weeks, as assessed by morphology (data not shown) In contrast, the remaining cells continued to proliferate over the weeks and formed colonies that were distinguishable under light-field microscopy (data not shown) tsA58T Ag-negative senescent cells were progressively excluded by serial passages At day 30, the dishes consisted almost exclusively of viable tsA58T Ag-positive cells (Figs and 3A) Cells obtained from T26 single-trans- Fig Endothelial cell culture from organs of T26 ⁄ Tie2–Cre double-transgenic mice (A) Proliferating cells obtained from the brain were immunostained for SV40T Ag Proliferating cells without undergoing senescence were tsA58T Ag-positive DAPI, 4,6-diamidino-2-phenylindole (B,C) Immunostaining revealed that tsA58T Ag-positive proliferating cells obtained from each organ maintained expression of the endothelial-specific markers CD31 (B) and Lyve-1 (C) (D) 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI)-labeled acetylated LDLs are taken up by these cells Bar = 50 lm (D, right panel) or 200 lm (all other panels) FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS 1991 A new method for mouse endothelial cell culture T Yamaguchi et al substantial proportion of the cell population from the uterus (Fig 3C) also stained positive for Lyve-1, indicating that cell populations obtained from these tissues were a mixture of blood vascular ECs (BECs) and LECs DiI-labeled acetylated LDLs were taken up by all types of cell populations (Fig 3D for brain and uterine ECs; data not shown for others), but not by nonendothelial NIH3T3 cells (data not shown), indicating that the cells maintained the physiological characteristic of acetylated-LDL uptake Lyve-1-positive liver sinusoidal endothelial cells in vitro Intriguingly, almost all of the cell population from the liver were also positive for Lyve-1 (Fig 3C), and Western blot analysis revealed that they were Lyve-1-positive, prospero-related homeobox-1 (Prox-1)-negative [34] ECs (Fig 4B), suggesting that the population represented Lyve-1-positive liver sinusoidal ECs [30–32] and maintained the property of Lyve-1 expression in vitro These results also suggest that Lyve-1 expression in liver sinusoidal ECs, reported as a marker of differentiated organ-specific ECs [32] and a potential diagnostic marker of liver cancer and cirrhosis [30], is regulated in a cell-autonomous manner and is irreversible in the culture conditions used in this study These cultured ECs might allow us to investigate more properties of liver sinusoidal ECs in health and disease ECs were enriched as expected Western blot analysis revealed that Prox-1 and vascular endothelial growth factor receptor (VEGFR-3), which is expressed predominantly in LECs [35,36], were also expressed in Lyve-1-positive ECs (Fig 4B), confirming that LECs were obtained from the mixed EC population tsA58T Ag-positive BECs and LECs transduced signals of endothelial growth factors We further examined whether isolated ECs constitutively expressing tsA58T Ag could respond to angiogenic and lymphangiogenic growth factors Serum-depleted LECs were treated with vascular endothelial growth factors A or C (VEGF-A or VEGF-C) (Fig 4C) Phosphorylation of VEGFR-2 and mitogenactivated protein kinases (MAPKs), but not of VEGFR-3, was induced by VEGF-A, whereas phosphorylation of VEGFR-2, VEGFR-3 and MAPKs was induced by VEGF-C, as reported in a previous study using human primary LECs [36] These results suggest that growth factor signals were transduced properly via endothelium-specific receptors in these cells Mesenteric BECs and LECs (Fig 5A,B) were also obtained by the same strategy as illustrated in Figs and 4, and were treated with VEGF-A or VEGF-C (Fig 5C) MAPK and Akt phosphorylation were induced in both BECs and LECs by stimulation with VEGF-A or VEGF-C, indicating that the cultured ECs responded to the endothelial growth factors Isolation and characterization of BECs and LECs We next isolated LECs from the mixed cell population by magnetic immunosorting using an antibody against Lyve-1 (Fig 4A) We used uterine ECs for this purpose because they contained large numbers of Lyve-1-positive cells as assessed by immunostaining (Fig 3C) and further confirmed by double staining for Lyve-1 and another lymphatic endothelial marker, Prox-1 [34] (Fig 4A) As shown by the immunostaining of positively sorted or depleted cells (Fig 4A), Lyve-1-positive Implications for tube formation-based assays and transfection assays of tsA58T Ag-expressing ECs We also examined whether the cells formed tube-like structures on collagen gel Both uterine BECs and LECs could form tube-like structures (Fig 4D) In addition, an SV40-ori-containing plasmid carrying a GFP expression cassette could be introduced by lipofection and maintained for at least days after transfection as assessed by GFP expression (Fig 4E) These Fig Isolation and characterization of uterine BECs and LECs expressing tsA58T Ag (A) Scheme for sorting of LECs from the uterine EC population (days 30–40) A substantial proportion of uterine ECs were positive for Lyve-1 and Prox-1 (red and green on the top panel, respectively), indicating that the uterine EC population was a mixed cell population of BECs and LECs Lyve-1-positive LECs were isolated from mixed ECs by magnet immunosorting using anti-Lyve-1 antibody Scale bars ¼ 200 nm (B) Western blotting revealed that Lyve-1-positive uterine ECs maintained expression of Lyve-1, Prox-1 and VEGFR-3, indicating that they represent LECs In contrast, liver ECs were positive for Lyve-1 but not for Prox-1, indicating that they represent liver sinusoidal ECs (C) The uterine LECs transduced growth-factor signals via VEGFR-2 and VEGFR-3 IP, immunoprecipitation; IB, immunoblot; P-Y, phosphotyrosine (D) The uterine BECs and LECs formed tube-like structures Bars = 200 lm (E) SV40-ori-positive plasmids bearing GFP and drug-resistance genes were maintained in the uterine BECs and LECs for at least days after transfection under drug-selection pressure Bars = 200 lm All cells were cultured at 33 °C, and day 40–50 ECs were used for experiments shown in B–E 1992 FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS VEGF-A C Uterine ECs No treatment DaAPI Lyve-1 Prox-1 A A new method for mouse endothelial cell culture VEGF-C T Yamaguchi et al 250 kDa Magnetic immunosorting using anti-Lyve-1 Ab IP: VEGFR-3 IB: P-Y 150 kDa 250 kDa IP: VEGFR-3 150 kDa 250 kDa IB: P-VEGFR-2 250 kDa IB: VEGFR-2 Negative CDa31 DaAPI Lyve-1 DaAPI Positive IB: VEGFR-3 50 kDa IB: P-MAPK 37 kDa 50 kDa IB: MAPK D Uterine ECs (Lyve-1–) BECs E 75 kDa 75 kDa Lyve-1 Prox-1 250 kDa 150 kDa BECs days 50 kDa 100 kDa LECs 24 h Uterine ECs (Lyve-1+) Lung ECs Liver ECs Heart ECs Brain ECs B Uterine ECs (unsorted) 37 kDa VEGFR-3 75 kDa / tublin days 50 kDa tsA58 T Ag 24 h LECs 100 kDa 100 kDa FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS 1993 BECs LECs B T Yamaguchi et al LECs A BECs A new method for mouse endothelial cell culture CD31 Prox-1 SV40T VEGFR-3 Lyve-1 SV40T VEGF-C VEGF-A No treatment VEGF-C VEGF-A C No treatment Lyve-1 The early mortality of the T26 ⁄ Tie2–Cre doubletransgenic mice is a disadvantage for the generation of T26 ⁄ Tie2–Cre double-transgenic mice with a mutation in a gene-of-interest In addition, non-ECs expressing tsA58T Ag derived from Tie2-expressing hematopoietic cells and embryonic endothelial cells may be contaminants in the present culture system In order to overcome these disadvantages, the use of tamoxifen-inducible Cre-expressing mice, such as the VE–cadherin–CreERT2 mice [37], may be preferable Homozygous T26 transgenic mice may also facilitate production of these animals, although it remains to be determined whether the homozygous mice are viable and fertile Experimental procedures p-MAPK Mice C57BL ⁄ 6J mice and MCH:ICR mice were purchased from CLEA Japan (Tokyo, Japan) Tie2–Cre transgenic mice (B6.Cg-Tg(Tek-cre)12Flv ⁄ J, #004128) [27] were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) All mice were housed under pathogen-free conditions All of the work with mice conformed to guidelines approved by the Institutional Animal Care and Use Committee of the University of Tokyo MAPK p-Akt Akt BECs LECs Fig Isolation and characterization of mesenteric BECs and LECs expressing tsA58T Ag Mesenteric ECs were obtained from an 8-week-old T26 ⁄ Tie2–Cre double-transgenic mouse as illustrated in Fig (A) CD31-positive, Lyve-1-negative BECs and CD31-positive, Lyve-1-positive LECs expressing tsA58T Ag were separated by magnetic immunosorting using anti-Lyve-1 antibody by the method illustrated in Fig 4A Red, Lyve–1 and CD31; green, SV40T Ag Bar = 200 lm (B) Prox–1 and VEGFR–3 were also expressed in Lyve–1-positive ECs, confirming that they maintained lymphatic endothelial properties (C) VEGF–A and VEGF–C induced MAPK and Akt activation in both populations of mesenteric ECs Day 30–40 endothelial cells were cultured at 33 °C and used in (B) and (C) results suggest that these cells can not only be used in functional analyses based on tube-like formation, but also used in gain-of-function, knockdown or rescue analyses using expression vectors Taken together, these results demonstrate that tsA58T Ag-positive BECs and LECs can be isolated by a simple method using our transgenic system and maintained at 33 °C without overt alterations in endothelial properties, including specific gene expression, physiological functions and intracellular signaling Thus, our system provides an accessible method to examine the endothelial cell biology of the mouse, and will accelerate the molecular and cellular analysis of ECs and their heterogeneity in various vascular beds 1994 Construction of the transgene Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) from COS-7 cells harboring the wild-type SV40T Ag gene (purchased from Health Science Research Resources Bank, Osaka, Japan) The RNA was reverse-transcribed using SuperScript II (Invitrogen) and used for cloning the SV40T Ag cDNA The cDNA encoding the wild-type SV40T Ag and the 3¢ portion of the tsA58T Ag cDNA carrying the A438V mutation were PCR-amplified from COS-7 cDNAs using the following primers: LTA-1F, 5¢-CTC GAGATGGATAAAGTTTTAAACAGAG-3¢ and LTA1R, 5¢-TGAAGGCAAATCTCTGGAC-3¢ for the former, and LTA–M2F, 5¢-CAGCTGTTTTGCTTGAATTATG-3¢ and LTA–2R, 5¢-GAATTCATTATGTTTCAGGTTCA GGGG-3¢ for the latter The PCR products were cloned into the EcoRV site of pZErO-2 (Invitrogen) A XhoI–PvuIIdigested fragment of the wild-type SV40T Ag cDNA and a PvuII–EcoRI-digested fragment of the ts58T Ag cDNA were re-ligated and subcloned into XhoI–EcoRI-digested pZErO-2 and sequence-verified The pCGX vector was constructed by replacing the EcoRI–HindIII fragment of pCAGGS [25] (kindly provided by J.-I Miyazaki, Osaka University, Japan) with the following fragments: b–geo cDNA with the polyadenylation signal sequence of the bovine growth hormone gene derived from pSA–bgeo [26] (kindly provided by H Niwa, RIKEN Center for Developmental Biology, FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS T Yamaguchi et al Kobe, Japan), two synthetic lox P sequences and cloning sites, a polyadenylation signal sequence of the mouse Pgk gene derived from pGT-N28 (NEB, Ipswich, MA, USA), and a portion of the multiple cloning site derived from pMCS5 (MoBiTec, Goettingen, Germany) Briefly, the lox P-flanked b–geo cassette was cloned under the control of the CAG promoter, followed by several cloning sites including a SwaI site, polyadenylation signal sequence of the Pgk gene, and a portion of the multiple cloning site of pMCS5 XhoI–EcoRI-digested tsA58T Ag cDNA was blunted and cloned into the SwaI site of pCGX, and the direction was verified by enzymatic digestion and sequencing Generation of a transgenic mouse line carrying the CAG–b–geo–tsA58T Ag transgene SalI-digested pCGX harboring the tsA58T Ag cDNA was resolved by electrophoresis, and a plasmid vector-free fragment was electro-eluted, phenol-extracted, ethanol-precipitated, and dissolved in NaCl ⁄ Pi A 10 lg aliquot of the transgene was introduced into E14.1 ESCs by electroporation ESCs expressing the transgene were selected by incubation for days in medium containing a concentration of G418 (Invitrogen) of 400 lgỈmL)1 G418-resistant colonies were picked and expanded for PCR genotyping and the formation of embryoid bodies One ESC clone (T26) out of 48 G418-resistant clones was further examined for the presence of the tsA58T cDNA expression unit and for widespread expression of b–geo in embryoid bodies To validate binary expression of the transgene, a plasmid vector harboring CAG–Cre (kindly provided by I Saito, Institute of Medical Science, University of Tokyo, Japan) was introduced into T26 ESCs by electroporation The resulting b–geo-free subclones (T26d) were selected and propagated for Western blot analysis of the SV40T Ag For production of transgenic mice, T26 ESCs were injected into B6 blastocysts The resulting blastocysts were transplanted into the uterus of pseudo-pregnant MCH:ICR female mice Chimeric male mice were then crossed with B6 female mice T26 transgenic mice were back-crossed five times or more with B6 mice and used for analysis For genotyping, PCR and ⁄ or X-gal staining of tail tips were performed ESCs were maintained on a layer of irradiated, G418resistant mouse primary embryonic fibroblasts in highglucose (4.5 gỈL)1) Dulbecco’s modified Eagle’s medium supplemented with 15% fetal bovine serum, 0.1 mm 2-mercaptoethanol and a culture supernatant of leukemia inhibitory factor (LIF)-producing BMT10 cells (kindly provided by J I Miyazaki, Osaka University, Japan) Embryoid bodies were obtained by culturing ESCs in the medium not supplemented with LIF-conditioned medium on non-coated dishes A new method for mouse endothelial cell culture Immunohistochemistry and immunocytochemistry Embryos and tissues were collected, fixed in 4% paraformaldehyde (PFA) overnight at °C, processed in NaCl ⁄ Pi containing 20% sucrose, and embedded in OCT (optimum cutting temperature) compound (Sakura Finetec, Tokyo, Japan) Sections (10–15 lm) of several tissues were cut using a cryotome (Sakura Finetech) The sections were mounted onto Matsunami adhesive silane-coated slides (Matsunami, Osaka, Japan) and dried overnight at room temperature The dried specimens were rehydrated in NaCl ⁄ Pi and then antigen-retrieved for the detection of SV40T Ag by incubation in NaCl ⁄ Pi containing 0.1– 0.25% trypsin and 0.5 mm EDTA at 37 °C or room temperature for 10–25 Prior to incubation with primary antibodies, all sections were incubated in NaCl ⁄ Pi or methanol containing 3% H2O2 at room temperature for 10–15 The primary antibodies used in this study were as follows: anti-PECAM-1 (BD Pharmingen, Franklin Lakes, NJ, USA), anti-Lyve-1 (R&D, Minneapolis, MN, USA), anti-Prox-1 (Acris Antibodies, Hiddenhausen, Germany), and anti-SV40 T Ag (Santa Cruz Biotechnology, Santa Cruz, CA, USA) The corresponding secondary antibodies labeled with horseradish peroxidase (HRP) (Biosource, Invitrogen), AlexaFluor 488 or AlexaFluor 546 (Molecular Probes, Invitrogen) were used Alternatively, Histofine (Nichirei Biosciences, Tokyo, Japan) was used For double immunostaining using HRP-conjugated antibodies for both of the secondary antibodies, sections stained with the first primary and secondary antibody were incubated in NaCl ⁄ Pi containing 3% H2O2 at room temperature for 15 prior to incubation with the second primary antibody The signal-enhancing TSA Plus fluorescence system (Perkin-Elmer, Waltham, MA, USA) for HRP-conjugated secondary antibodies was also used for visualization 4,6-diamidino-2-phenylindole (Molecular Probes) was used for nuclear staining Fluorescent microscopic photographs were acquired using an Olympus IX70 microscope with DP70 imaging system (Olympus, Tokyo, Japan) For immunocytochemistry, cells were fixed on ice with 4% PFA in NaCl ⁄ Pi for 10 min, incubated in methanol at )20 °C for 20 min, and rehydrated in NaCl ⁄ Pi For detection of Prox-1, cells were further bleached and the TSA Plus fluorescence system was used For the detection of other proteins, AlexaFluor-conjugated secondary antibodies were used for visualization Western blot analysis Cell lysates (40 lg, or 20 lg for lysates from cells shown in Fig 5) were loaded, resolved by SDS–PAGE, and wet- or semi-dry-blotted onto poly(vinylidene difluoride) FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS 1995 A new method for mouse endothelial cell culture T Yamaguchi et al membranes (Bio-Rad, Hercules, CA, USA) Western blot analysis was performed using the following primary antibodies: goat anti-Lyve-1 polyclonal IgG (1 : 500; Santa Cruz), rabbit anti-Prox-1 polyclonal IgG (1 : 500; Upstate ⁄ Millipore, Billerica, MA, USA), rat anti-VEGFR-3 monoclonal IgG (AFL4, : 500; eBioscience, San Diego, CA, USA), rat anti-VEGFR-2 monoclonal IgG (Avas2a, : 500; eBioscience), rabbit phospho-VEGFR-2 monoclonal IgG (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-SV40 large T antigen polyclonal IgG (1 : 1000; Santa Cruz), rabbit anti-a ⁄ b-tubulin polyclonal IgG (1 : 1000; Cell Signaling Technology), rabbit anti-phosphop42 ⁄ 44 MAPK polyclonal IgG (1 : 1000, Cell Signaling Technology), rabbit anti-p42 ⁄ 44 MAPK polyclonal IgG (1 : 1000, Cell Signaling Technology), rabbit anti-phospho– Akt polyclonal IgG (1 : 1000, Cell Signaling Technology) and anti-Akt polyclonal IgG (1 : 1000, Cell Signaling Technology) The secondary antibodies were swine antigoat IgG (HRP) (1 : 1000; Biosource), goat anti-rabbit IgG (HRP) (1 : 1000; Cell Signaling Technology or GE Healthcare, Piscataway, NJ, USA; : 2000 used for the detection of rabbit primary antibodies purchased from Cell Signaling Technology), and goat anti-rat IgG (HRP) (1:1000; Biosource) Skim milk (5%) in Tris-buffered saline containing 0.05-0.1% Tween 20 was used for blocking nonspecific antibody binding Antibody-labeled bands were visualized using an enhanced chemiluminescence kit (GE Healthcare) and X-ray film (Fujifilm, Tokyo, Japan) To detect phosphorylated tyrosine residues in VEGFR3, rabbit anti-VEGFR-3 polyclonal IgG (Santa Cruz) and protein G (Calbiochem ⁄ Merck, Darmstadt, Germany) were used for immunoprecipitation Blocking One-P (Nakarai-tesque, Kyoto, Japan) was used for blocking The membranes were incubated with mouse anti-phosphotyrosine monoclonal IgG (4G10, : 2000; Upstate) at °C overnight, followed by incubation with sheep anti-mouse IgG (HRP) (1 : 4000; GE Healthcare) at room temperature for h ‘Can Get Signal’ solution (Toyobo, Osaka, Japan) was used for dilution of both antibodies Cell culture and isolation of lymphatic endothelial cells from an endothelial-cell population Tissues were collected, washed in NaCl ⁄ Pi, and dissociated by agitation in Hanks’ balanced salt solution containing 0.2% type IV collagenase or NaCl ⁄ Pi containing 0.1% trypsin for 30–60 at 37 °C After pipetting the solution containing digested tissues several times, the enzyme-containing buffer was thoroughly removed by centrifugation and washing several times with NaCl ⁄ Pi Dissociated cells were filtered through a 100 lm nylon mesh to remove undissociated tissues, and cultured in microvascular endothelial cell medium (EGM-2MV) (Lonza, Basel, Switzerland) at 33 °C, the permissive temperature for 1996 tsA58T Ag The initial cells attached to dishes were passaged when sub-confluent to remove dead cells and small pieces of tissues Confluent cells were usually passaged every 2–3 days at a split ratio of : 3, but several passages around day 20 were performed without splitting because tsA58T-negative cells had undergone senescence, decreasing the cell number Isolation of Lyve-1-positive endothelial cells was performed using magnetic immunosorting Magnetic-activated cell separation (MACS) columns and MACS goat anti-rat IgG microbeads (Miltenyi Biotec, Bergisch Galdbach, Germany) were used according to the manufacturer’s protocol Attached cells were trypsinized, collected and counted Cells (1 · 107) were resuspended with 50 lL of MACS buffer containing 50 lgỈmL)1 antibody against Lyve-1 (MAB2125, R&D) and incubated on ice for 5–10 The primary antibody-labeled cells were washed twice with 500–1000 lL MACS buffer, resuspended in 100 lL MACS buffer containing microbeads, and incubated on ice for 15 Following a rinse with MACS buffer, the cells were suspended with 500 lL MACS buffer and applied onto MACS columns Magnetically selected positive cells and depleted cells were cultured independently as lymphatic endothelial cells and blood vascular endothelial cells, respectively Prior to activation with growth factors, cells were cultured in endothelial cell basal medium (EBM-2) basal medium (Cambrex) without either serum or supplemental growth factors for 16–18 h Recombinant human VEGF-A (Peprotech EC, London, UK) or rat VEGF-C (R&D) was added to EBM medium at a final concentration of 100 ngỈmL)1 Cells were then incubated at 33 °C for 20 and harvested for analysis For the experiment shown in Fig 5, mesenteric endothelial cells were isolated by the method described above with slight modification Prior to treatment with growth factors, cells were cultured in EBM-2 basal medium with 0.5% serum for 24 h Cells were incubated with growth factors at 33 °C for 10 and harvested for analysis Tube formation of tsA58T-expressing endothelial cells on collagen gel Matrigel (9.5 mgỈmL)1; BD Pharmingen) was placed into 24-well dishes, and 2–4 · 104 endothelial cells were seeded on the Matrigel and cultured for 24 h at 33 °C Uptake of DiI-labeled acetylated LDL into tsA58T-expressing endothelial cells DiI-labeled acetylated LDL was added to the culture medium at a final concentration of 10 lgỈmL)1, and the cells were incubated at 37 °C overnight Dishes were washed with NaCl ⁄ Pi and observed by fluorescent microscopy FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS T Yamaguchi et al NIH3T3 cells (Health Science Research Resources Bank, Osaka, Japan) were used as a negative control Transfection of plasmids harboring a GFP-expressing cassette into tsA58T-expressing endothelial cells Transfection was performed using Lipofectamine LTX and pcDNAÔ6.2-GW ⁄ miR-neg control plasmid (Invitrogen) according to the manufacturer’s protocol GFP signals were used to assess transfection and expression of the plasmid Acknowledgements We thank Dr Jun-ichi Miyazaki (Osaka University, Japan) for providing pCAGGS, pCAG-LIF and BMT10 cells; Dr Hitoshi Niwa (RIKEN Center for Developmental Biology, Kobe, Japan) for providing pSA-bgeo; Dr Izumo Saito (Institute of Medical Science, University of Tokyo, Japan) for providing pCAG-Cre; Dr Ikuo Yana (Institute of Medical Science, University of Tokyo, Japan) for help in performing endothelial tube formation experiments This work was supported by grants from the Japan Society for the Promotion of Science (to T I.) and the Ministry of Education, Culture, Sports, Science and Technology, Japan (to T Y., T I., N Y and H I.) 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GAGATGGATAAAGTTTTAAACAGAG-3¢ and LTA1R, 5¢-TGAAGGCAAATCTCTGGAC-3¢ for the former, and LTA–M2F, 5¢-CAGCTGTTTTGCTTGAATTATG-3¢ and LTA–2R, 5¢-GAATTCATTATGTTTCAGGTTCA GGGG-3¢ for the latter The PCR products... functions and intracellular signaling Thus, our system provides an accessible method to examine the endothelial cell biology of the mouse, and will accelerate the molecular and cellular analysis of. .. Heart Yolk sac Brain Embryo A chemistry using antibodies against the pan-EC marker, CD31, the lymphatic endothelial and liver sinusoidal endothelial marker Lyve-1 (lymphatic vessel endothelial