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Fig. 1. Targeted expression of human SPRY2 and EGFP in the
ureteric bud in vivo. (A) Schematic structure of the expression construct
generated by inserting a PCR fragment of human SPRY2 cDNA into the
EcoRI and BamHI sites of a plasmid containing
IRES-EGFP. The fragment was excised with EcoRI and
SspI, and inserted into the EcoRI and SmaI sites of
a modified bluescript vector. A ß-globin splice acceptor was
then cut with BamHI and EcoRV, blunted and inserted into a
blunted NotI site (N) downstream of the Pax2 promoter. The
fragment containing the Pax2 promotor and ß-globin was
removed from the vector with EcoRI and ligated upstream of the human
SPRY2 cDNA, IRES EGFP-PA. The yellow arrow indicates the
start site of transcription. B, BamHI; EcoRI site, (N)
defective NotI site; E, EcoRI. (B) An expected 300 bp
fragment was amplified with P1 and P2 primers in PCR, indicating the presence
of the transgene in the genome of the carriers. (C) The copy number of the
transgene was estimated by Southern blotting using GFP as a probe.
Control of loading is indicated below. The Pax2 promoter drives
expression of EGFP in the ureteric bud of a kidney at E11.5 (D) and
E17.5 (F). The human SPRY2 gene is also expressed in the kidney at
E12.5 (E) and E17.5 (G), as judged by in situ hybridization. WT, wild type;
TG, transgene carrier. Scale bars: 100 µm.
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