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Figure 1 | BMC Developmental Biology

Figure 1

From: An in vivo reporter of BMP signaling in organogenesis reveals targets in the developing kidney

Figure 1

Strategy for generating the BRE-lacZ reporter strain. A. The BRE-lacZ transgene consists of a duplex of the CAGC1/2 sequence, and a duplex of SMAD binding elements 2 and 3 derived from the mouse Id1 gene [15] upstream of the Hspa1a promoter fused to β-galactosidase and the SV40 polyadenylation signal [27, 28]. B. ES cell transgenesis was used to facilitate screening for signal strength of the reporter: ES cells were co-electroporated with BRE-Hspa1a-lacZ and a pgk-neo drug selection cassette, drug resistant colonies were isolated and screened for strong β-galactosidase expression in response to treatment with 25 ng/ml BMP4. Three clones were selected for blastocyst injection. C. Responsiveness of murine embryonic fibroblasts (MEFs) derived from BRE-lacZ positive E12.5 embryos was assessed by X-gal staining of cells. D. Responsiveness of MEFs to BMP treatment was verified by quantitative assay of β-galactosidase activity using the ONPG substrate. E. BRE-lacZ MEFs cultured in serum free medium for 48 hours with and without the addition of noggin were assayed for β-galactosidase activity using the ONPG substrate. For comparison of BMP pathway activation, cell extracts were immunoblotted for pSMAD1/5/8. Comparable loading of protein on the membrane was verified by immunoblot with β-tubulin.

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