Generation of mWisp3 cDNA Full-length mWisp3 cDNA (Genbank accession number: XP_282903) was amplified by RT-PCR using mouse testis cDNA and cloned into the Not I and Xba I sites of pcDNA3.1 vector (Invitrogen). Constructs were sequenced to verify that no PCR-induced mutations had occurred and there was no difference between the mWisp3 clone and the Genbank sequence.
Transient transfection of HEK293T cells with the mWisp3 expression vectors used to produce transgenic mice HEK293T cells were grown in Dulbecco’s Minimum Essential Medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PSM) (Invitrogen). Cells were maintained in 10 cm tissue culture plates (Falcon) at 37°C with 5% CO2 in a humidified incubator. HEK293T cells were transfected at 70–80% confluency with 4 μg of empty expression vector or vector containing mWisp3 cDNA using LipofectAmine/Plus Reagent (Invitrogen) in 6.5 mL serum-free DMEM. Four hours (h) after transfection, 6.5 mL of DMEM supplemented with 20% FBS was added to the cultures and allowed to incubate overnight. The medium was then replaced with 10 mL of DMEM containing 10% FBS for 24 h followed by 5 mL of serum-free DMEM for 24 h. Conditioned medium was then recovered and centrifuged at 16,000 g for 5 min. One-hundred-twenty-five μL of supernatant was then acetone precipitated (5:1 acetone:medium) overnight at −20°C followed by centrifugation at 16,000 g for 5 min. Acetone was poured off and the pellets were dried before re-suspension in 40 μL 1× sodium dodecyl sulfate (SDS) sample loading buffer at 55°C for 10 min. Cell lysate was recovered by scraping following incubation with 1 mL RIPA buffer (150 mM NaCl, 0.5% deoxycholate, 1% NP-40, 0.1% SDS, and 50 mM Tris, pH 8.0) for 5 min. Cell lysate was centrifuged at 16,000 g for 5 min to clear insoluble cellular debris. Equal volumes of supernatant and 2 × SDS sample loading solution were then mixed. β-mercaptoethanol was added to cell lysate and conditioned medium samples (5% v/v) prior to boiling and separation by SDS-PAGE.
Generation of mWisp3 transgenic mice
The plasmid vector pcDNA3.1, which drives transgene expression in multiple tissues via a CMV promoter (Yu et al. 2005
; Valverde et al. 2008
), was used to produce one transgenic strain of mice (CMV-WISP3). The pCAGGS vector, which drives transgene expression using a chicken-β-actin promoter/CMV enhancer (Niwa et al. 1991
), was used to produce another transgenic strain (β-actin-WISP3). Full-length mWisp3
cDNA was subcloned into the EcoRI
site of pCAGGS vector. Plasmid DNA was injected into the pronuclei of B6-SJL/F2 oocytes and placed into pseudopregnant females to produce transgenic offspring. Transgenic animals were identified by Southern blot using tail DNA with a mWisp3
cDNA probe. Transgenic animals were then mated with 129/SvEv wild-type mice to test for germline transmission of the transgene.
Characterization of mWisp3 transgenic mice Mice were maintained in a specific pathogen free facility and permitted to age with their non-transgenic littermates. Mice were inspected daily for phenotypic findings. Mice were sacrificed and processed for RNA and protein extraction, primary cell culture, and histologic examination at time points ranging from embryonic day 14 (E14) to 62 weeks-of-age.RT-PCR was performed using total RNA from fresh-frozen tissue samples after extraction using Isogen (Nippon Gene Co.) according to the manufacturer’s instructions. RT-PCR product was subcloned and sequenced using standard methods.Histologic specimens were fixed in 10% buffered formalin (Fisher) before being placed in Tissue-Tek biopsy cassettes (Sakura Finetek). Samples were then processed and embedded in paraffin. Five μm-thick tissue sections were collected on Superfrost/Plus slides (Fisher). Images were viewed on a Leica DMLB microscope (Leica Microsystems Inc.) and captured using a Spot Camera and software (Diagnostics Instruments, Inc.).For Western blotting, entire hearts, kidneys, or femoral articular cartilages were immediately frozen in a methanol/dry ice bath, homogenized in pestle tubes, and then extracted on ice for 20 min in 1 mL Triton X-100 buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 1% Triton X-100, 1 mM EGTA, 10% Glycerol, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM sodium vanadate, 50 mM sodium fluoride, and proteinase inhibitor mixture (Roche)). The protein concentrations were determined by Bradford Assay, and the samples were stored at −80°C until use.
Primary kidney cell culture
Four-week-old β-actin-WISP3 transgenic and non-transgenic littermates were sacrificed and their kidneys were recovered. After three washes with sterile PBS containing 10% PSM, the kidneys were minced into small pieces and digested in 10 mL DMEM/F12 medium containing 1 mg/mL Collagenase A (Roche) and 10% BSA on a 10 cm dish at 37°C for 30 min in a tissue culture incubator. Following gentle suspension using a pipet, the mixture was passed through a cell strainer into a 50 mL Falcon tube and centrifuged at 1,000 g for 10 min. The supernatant was discarded and the cell pellet was suspended in 3 mL of fresh DMEM/F12 containing 20% FCS and 1% PSM. From each transgenic and non-transgenic kidney 1
cells/mL were plated onto 10 cm tissue culture plates. The medium was changed daily for 2 days. Adherent cells were recovered by trypsin digestion, re-plated, and grown to confluence in another 2 days. Conditioned medium and cell lysate were then recovered, as described above, and stored −80°C until use.
Immunodetection of WISP3 following SDS-PAGE
Tissue extract, conditioned medium, and cell lysate samples were electrophoresed in 12% Tris-HCl gels (BioRad), transferred to Immobilon P membrane (Millipore Corp.), and then blocked with 4% dry milk in Tris-buffered saline (TBS) at 4°C overnight. Primary and secondary antibodies were diluted using 4% dry milk/TBS with 0.05% Tween 20 (TBST). WISP3-C rabbit polyclonal antibody (Kutz et al. 2005
; Nakamura et al. 2007
) was used as the primary antibody (1:500 dilution) and goat anti-rabbit HRP-conjugated antibody (Pierce) was used as the secondary antibody (1:5,000). ECL plus Western blotting Detection System (Amersham Biosciences) and X-OMAT AR film (Eastman Kodak Company) were used for developing blots and detecting signal.
Generation of expression constructs and in vitro transcription of RNAs for injection into zebrafish embryos
cloned into the pCS2+ expression vector has been described previously (Nakamura et al. 2007
). Full-length mWisp3
was also cloned into the pCS2+ vector. Other cDNA constructs used in this study included human DKK1 (hDKK1
) and zebrafish Wnt8 (zWnt8
) (Nakamura et al. 2007
). The RNAs were synthesized from Not
I digested pCS2+ plasmids using the sp6
mMessage mMachine kit (Ambion). Phenol red (0.1%) was added to the RNA solution as a tracer, and 150 pg of RNA was injected into 1 or 2-cell-stage embryos. Following injections, embryos were cultured in aquatic system water. Phenotypic scoring for evidence of BMP and Wnt inhibition were as previously described (Nakamura et al. 2007
COS7 cells were cultured in DMEM supplemented with 10% FBS. Cells were plated at 2
cells per well in six-well culture plates 24 h prior to transfection. Cells were co-transfected with 2 μg of mBMP4-myc plasmid and 0.5 μg of mSPC4 plasmid (Nakamura et al. 2007
). Twenty-four h after transfection, the culture medium was changed to 1 mL OPTIMEM (Gibco-BRL) containing 1% Knockout SR (Invitrogen
). mBMP4-myc-containing conditioned medium was collected 48 h later and stored frozen at −80°C until use. Five-hundred μL of mBMP4-myc-containing conditioned medium was mixed with either 1 mL of β-actin-WISP3-transgenic, or non-transgenic, primary kidney cell culture conditioned medium and 65 μL of anti-myc beads (Santa Cruz Biotechnology). The samples were rocked overnight at 4°C. Beads were pelleted, washed four times at 4°C for 5 min each in RIPA buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS), then resuspended in 65 μL 2 × SDS-PAGE loading buffer with 5% BME. After boiling for 5 min, 20 μL were separated by SDS-PAGE, transferred to Immobilon P and immunodetected using HRP-conjugated anti-myc antibody (Santa Cruz Biotechnology) and WISP3-C antibody. Immunoreactive bands were detected as previously described.
Bioassay for BMP signaling in mammalian cells
BMP signaling bioassays were performed using mouse C3H/10T1/2 cells. Cells were plated in 96-well plates (2
cells/well) and recombinant human BMP2 (rhBMP-2) (R&D Systems) was added to the culture medium (final concentration
100 ng/mL). Cells were then cultured in 50 μL of DMEM (10% FBS) and 50 μL of β-actin-WISP3-transgenic or non-transgenic primary kidney cell conditioned medium. The culture medium was changed every other day, until the cells were harvested on day 6. The cells were washed twice with ice-cold PBS and alkaline phosphatase activity was measured with the LabAssayTM
ALP kit (Wako Chemicals USA Inc.) according to the manufacturer’s protocol. Total protein content was determined with the BCA Protein Assay kit (Pierce Chemical Co.) using bovine albumin as a standard. Results are expressed as units ALP activity per microgram of total cell protein. Experiments were performed in triplicate on two separate occasions.