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For Plaque Specific Expression

In order to direct microglia activity to senile plaques , we needed to find a way to detect these plaques. Several possible routes were explored; initial focus was on a plaque binding protein, such as the B10 antibody (Haupt et al. 2011). However, there was no easy way for plaque binding to transduce changes in gene expression. Therefore, alternatives were explored, where plaque proximity could be indirectly detected via a proxy. One such proxy is oxidative stress - free radical production (Colton et al., 2000) which is generated by plaques. Microglia are naturally attracted to plaques, and upon reaching plaques, a standard immune response follows, which also includes free radical production. Therefore, we have designed a promoter which will initiate transcription in response to the oxidative stress generated by native microglia and plaques already present in the brain.

This promoter is an improvement of a yeast minimal promoter cyc1001 already in the registry. Although from yeast, parts of this promoter show homology to the consensus sequences of mammalian core promoter elements, notably the TATA box and initiator element (Sandelin et al. 2007). NF-κB is a transcription factor which translocates to the nucleus under oxidative stress (Shi et al., 2003), and binds to the sequence GGGAATTT (Park et al., 2009). Thus, by placing this site upstream of the yeast minimal promoter, we created a novel mammalian promoter which initiates transcription in response to oxidative stress.

To construct this, we firstly created a BioBrick which consists of 5 copies of the NF-κB binding site (5NFKB [link to parts page]). This was done using linkers - short overlapping primers were ordered, and allowed to anneal, and then ligated together.


Protocol: Preparation

Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use. Analysis with Tris-Tricine gels indicated that the vast majority of Aβ in this preparation (referred to as sAβ) was in the monomer form.

To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.

Protocol: Congo Red Assay

a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use.

b. For fAβ digestions (employing Congo red), 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.

c. After digestion, analyse the reaction by Congo red assay.