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The correct expression pattern must be extracted from the registered image before conducting further analysis.
Furthermore, it is known from studies with AG, STK, and SEP3 that introns can contain important regulatory elements that are required for the correct expression pattern of these MADS box genes [ 29- 31].
Therefore, a genomic fragment containing ∼2.5 kb of promoter sequence up to exon 2, including the complete intron 1 containing enhancer elements essential for the correct expression pattern, was isolated and cloned into pBSK (Meadows et al., 2009).
To determine the role of the two selected MYB and the two MYC sites in the regulation of the correct expression pattern we mutated each site individually and both MYB and both MYC sites together.
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Complex cis-regulatory sequences like the ones driving expression of the Drosophila even-skipped gene have proven difficult to design from existing knowledge, presumably due to the large number of protein protein interactions needed to drive the correct expression patterns of genes in multicellular organisms.
As expected, we mostly detected correct expression patterns.
This appears also to be the case for SEP3, because fusion of GFP to a 3.5 kb genomic fragment of SEP3 including upstream and intron sequences revealed correct expression patterns.
The integrity of nucleosomal organization around TSS is essential for the maintenance of the correct gene expression pattern in a given cell.
This assay allowed us to determine not only the qualitative functionality of the promoter sequences, i.e. the correct spatial expression pattern in petals, but also quantitatively assess the levels of expression.
Of these 141 genes, 94 are also identified by our analysis, and a further 36 show the correct differential expression pattern although it fails to reach statistical significance using the conservative Bonferroni correction for multiple testing.
IHC staining for K5, K10, loricrin and involucrin in C/EBPβff and IKOβ epidermis treated with 4OHT revealed the correct spatial expression patterns, however, similar to germline C/EBPβ knockout mice [26], we observed a modest decreased staining intensity for K10 (Fig. 1H).
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com