rBMP7 rescues Ngn2 mRNA expression in cultured E14.5 Bmp7-deficient cortical cells.
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RT-PCR analysis of serial 4-fold cDNA dilutions (1, ¼, 1/16, 1/64, 1/256 cDNA input) for Ngn2 and Pax6 in isolated E14.5 wild-type (wt) or Bmp7
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Cell culture and immunoblotting
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Cranial fibroblasts were isolated from E14.5 C57BL/6 mouse embryos.
- The mid-facial region was dissected from embryo heads and incubated in 0.05% trypsin-EDTA solution for 1 h at 37°C, after which the tissue was triturated by repeated aspiration.
- Cells were centrifuged, resuspended in Dulbecco's Minimal Essential Medium (DMEM;) containing 10% fetal calf serum (FCS), plated onto 10 cm cell culture dishes, and incubated at 37°C with 6% CO2.
- At confluency, cranial fibroblasts were passaged by trypsinization.
- Bmp7 stimulation experiments were performed in 24-well dishes with cells from the second passage.
- Fibroblasts were first starved for 24 h in DMEM/0.3% FCS, before the medium was changed to DMEM/0.3% FCS containing human recombinant Bmp7 (, , 0, 10, 20, 50, and 100 ng/ml, respectively).
- For positive controls, wells were incubated with DMEM/10% FCS.
- Cell supernatant was collected from individual wells after 24 h, run on…
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Cranial fibroblasts were isolated from E14.5 C57BL/6 mouse embryos.
- The mid-facial region was dissected from embryo heads and incubated in 0.05% trypsin-EDTA solution for 1 h at 37°C, after which the tissue was triturated by repeated aspiration.
- Cells were centrifuged, resuspended in Dulbecco's Minimal Essential Medium (DMEM;) containing 10% fetal calf serum (FCS), plated onto 10 cm cell culture dishes, and incubated at 37°C with 6% CO2.
- At confluency, cranial fibroblasts were passaged by trypsinization.
- Bmp7 stimulation experiments were performed in 24-well dishes with cells from the second passage.
- Fibroblasts were first starved for 24 h in DMEM/0.3% FCS, before the medium was changed to DMEM/0.3% FCS containing human recombinant Bmp7 (, , 0, 10, 20, 50, and 100 ng/ml, respectively).
- For positive controls, wells were incubated with DMEM/10% FCS.
- Cell supernatant was collected from individual wells after 24 h, run on SDS-7.5% polyacrylamide gels under reducing conditions, and proteins were transferred to nitrocellulose.
- Blots were briefly stained with 0.1% Ponceau Red, and then incubated with rabbit anti-mouse tenascin-W antiserum (see above) or rat anti-mouse tenascin-C monoclonal antibody mTn12 (obtained from Ruth Chiquet-Ehrismann and
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Eggs and Embryo Manipulations
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Fertilized White Leghorn chicken eggs were incubated at 38°C and the embryos staged according to Hamburger and in situ hybridization.
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Specification of intermediate mesoderm (IM) from the hESC-derived PS cells.
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(b) Comparison of OSR1 transcripts by combinatorial treatments with exogenous growth factors of retinoic acid , BMP7 (B7) and FGF2 (F2).
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