Figure 4

Expression of the endogenous E11 and E1 and thetransgene mRNAs in the transgenic mice during ontogeny. A. RT-PCR:The temporal expression of a 184 bp exons 1/2 (Exon 1/2) and a 178 bp exon 11 (Exon 11) transcripts during ontogeny of transgenic mice was determined by the same relative quantitative RT-PCR approach used in C57BL/6J mice. A 0.95 kb Exon 1/tau transcript and a 0.9 kb Exon 11/tau transcript were determined by a similar RT-PCR approach using the same first-strand cDNAs as templates, as described in the Methods section. RNA input was estimated by a parallel PCR with a pair of G3PDH primers. Agarose gel stained with ethidium bromide was photographed with FluorChem 8000 system. PCR cycles and extension time at 72°C: for Exon 1/2 primer set, 35 cycles and 20 sec; for Exon 11 primer set, 45 cycles and 20 sec; for Exon 1/tau primer set, 35 cycles and 2 min; for Exon 11/tau primer set, 45 cycles and 2 min, and for G3PDH primer set, 25 cycles and 2 min. B. Relative quantification: Relative band intensities from ethidium bromide-stained gel were quantified with AlphaEase FC software, and converted to amole concentration based upon the linear regression equations from the linear phase of each saturation curve (see Fig. 8) using Prism 4.0. The concentration was normalized with the G3PDH concentration. Right Y axis represents the concentration of Exon 11 and Exon11/tau cDNAs, and left Y axis, the concentration of Exon 1/2 and Exon 1/tau cDNAs. Bars represent the mean ± S.E. of the concentration from three independent experiments.