Expression patterns of DD RT-PCR clones and Wnt signaling members.
(A) RT-PCR analysis was performed to verify the differential expression of several clones derived from the DD RT-PCR screening. RNA isolated from AGM and fetal liver at E10, E11, and E12 was used for cDNA synthesis and RT-PCR under standard conditions. For each primer set, optimal PCR conditions and annealing temperature were determined and PCR was performed for 30 to 40 cycles. Three of the genes,mTAB2, mTM9SF2, and β-catenin (Figure 1B) are up-regulated between E10 and E11 in the AGM. The other genes(BUB1B and Cu/Zn SOD), not showing a differential expression pattern, represent false-positive DD RT-PCR clones. (B) RT-PCR analysis was performed for several components of the Wnt signaling pathway. Several components of the Wnt signaling pathway can be detected in the AGM and fetal liver between E10 and E12. Besides the differential expression of β-catenin between E10 and E11 in the AGM, we could also detect up-regulation of Dvl3and Wnt5 in the AGM between E10 and E11. + and − indicate with and without reverse transcriptase, respectively; H, H2O.
