The resulting Math1 cDNA was subcloned into Bam HI and Cla I sites of the Tet-On gene regulation vector pTRE2pur (plasmidr-Math1 plaSupplementaryentary Information, Supplementary Figure S1A).
f x, k,γ) denotes the regulation vector of synthetic gene network, which is the function of production rate parameters k and decay rate parameters γ to be designed.
Two down-regulation vectors were constructed based on Pogostick-up-1 and Pogostick-up-2.
Pogostick was designed to facilitate cloning of candidate genes to produce either over-expression or down-regulation vectors.
Subsequently, other over-expression and down-regulation vectors were also constructed using the same cloning strategy, suggesting its wide applicability for different genes.
And down-regulation vectors were constructed with CD fragments of wingless (459 bp, unpublished), decapentaplegic (437 bp, unpublished) and spalt (140 bp, unpublished) of B. anynana.
The cloning strategy outlined above, using only XbaI, NheI, SpeI or AvrII REs was successfully applied to construct DsRed and Ubx over-expression vectors using both of the Pogostick MCS (Pogostick-up-1, Pogostick-up-2) and DsRed down-regulation vectors (Pogostick-down-FR and Pogostick-down-RF).
For the construction of down-regulation vectors, a cDNA fragment of the target gene should be inserted into both MCS sites, in reverse orientation, to form inverted repeats (IR), hairpin RNA structures that can specifically silence gene expression via the mechanism of RNAi.
For down-regulation vectors, the size of the cDNA fragment should be between 500-1000 bp in length, as this triggers stronger silencing than shorter fragments [41] and should preferably be a single complete exon from the target gene, since exons often contain sequences that facilitate the processing of transcripts [41].
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