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Fig. 2. Injection of the Wt1 expression vector. (A) Effect on branching
morphogenesis. (a,c,d) Injection of Wt1 cDNA. (b,e) Injection of GFP vector.
In situ hybridization with Wt1 antisense probe (a,b), or sense probe
(c). The strong area of hybridization is at the single injection site (a), and
the endogenous Wt1 signal is observed at several induced nephrons (a,b). (d,e)
Cytokeratin staining demonstrating effect of Wt1 on branching. In experiments
designed to observe an effect on branching, the vector is injected at two
sites, one on either side of the ureteric bud. The branching comparisons shown
in each figure are between organ cultures obtained from the same embryo. (f)
Overall quantitation of 12 pairs of experiments, represented by d,e.
Significant differences in branching were detected after Wt1
injection (P<0.001). (B) Wt1 stimulation of
Vegfa expression. Injection of Wt1 (a,c) and Gfp
(b) expression vectors. In situ hybridization with Vegfa antisense
(a,b) and sense (c) probes. (d) Real-time PCR with Vegfa primers in
response to microinjections. As described in the text, a Gfp
expression vector was co-injected, and areas of Gfp expression were
separated from the remainder of the organ culture. The expression vectors used
are shown at the bottom of each column. Increased Vegfa mRNA is
observed in response to injection of a Wt1 expression vector. All
Vegfa Ct values were normalized to the Gapdh Ct value from real-time
PCR for the injected tissue.
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