Team:UCL/Project/Detection

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<p class="minor_title">IGEM: Intelligently Genetically Engineered Microglia</p>
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In order to direct microglia activity to senile plaques [internal link to neuropathology], we needed to find a way to detect plaques. Several possible routes were explored; initial focus was on a plaque binding protein, such as the B10 antibody (Haupt et al., 2011 [http://www.ncbi.nlm.nih.gov/pubmed/21059358]). 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 [http://www.ncbi.nlm.nih.gov/pubmed/10863548]); microglia are naturally attracted to plaques, and upon reaching plaques, a standard immune response follows, which 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.<a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">
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This promoter is an improvement of a yeast minimal promoter (cyc100 [http://parts.igem.org/Part:BBa_K105027]) already in the registry. Although from yeast, this 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 [http://www.ncbi.nlm.nih.gov/pubmed/17486122]). NF-κB is a transcription factor which translocates to the nucleus under oxidative stress (Shi et al., 2003 [http://www.ncbi.nlm.nih.gov/pubmed/12730877]), and binds to the sequence GGGAATTT (Park et al., 2009 [http://www.ncbi.nlm.nih.gov/pubmed/19435890]). 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.
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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 [link to experiments, linker construction] - short overlapping primers were ordered, and allowed to anneal, and then ligated together.
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Zeocin is a glycopeptide antibiotic capable of killing most bacteria, fungi, yeast, plant, and animal cells by intercalating DNA and inducing double strand breakage. This makes zeocin resistance an ideal selective maker for our project, which involves both bacterial and mammalian chassis. The product of the ''Sh ble'' gene, isolated from the bacterium Streptoalloteichus hindustanus <a href="http://www.ncbi.nlm.nih.gov/pubmed/2450783" target="_blank">(Gatignol et al. 1988)</a>, confers zeocin resistance to transfected/transformed cells. Sh ble is a small binding protein with strong affinity for antibiotics on a one to one ratio. It prevents zeocin from being activated by ferrous ions and oxygen, meaning it cannot react in vitro with DNA.
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Oxidative stress via free radical production increases with proximity to senile plaques <a href="http://www.ncbi.nlm.nih.gov/pubmed/10863548" target="_blank">(Colton et al., 2000)</a>. The microglia immune response around plaques also increases oxidative stress. Therefore, we have designed a promoter which will initiate transcription in response to oxidative stress to ensure the production of key proteins only in the plaques’ locales. This promoter is an improvement of a yeast minimal promoter (<a href="http://parts.igem.org/Part:BBa_K105027" target="_blank">cyc100</a>) already in the registry. NF-κB is a transcription factor which translocates to the nucleus under oxidative stress <a href="http://www.ncbi.nlm.nih.gov/pubmed/12730877" target="_blank">(Shi et al., 2003)</a>, and binds to the sequence GGGAATTT <a href="http://www.ncbi.nlm.nih.gov/pubmed/19435890" target="_blank">(Park et al., 2009)</a>. 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.
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MMP-9, also known as gelatinase B, is most commonly known for its role in breaking down the extracellular matrix. Naturally it is secreted in its inactive form and must be cleaved by other proteases, but our GEM are are meant to produce just the active form. It has been shown by Yan et al. that MMP-9 is the only known endogenous protease that degrades both fibrillar and soluble forms of amyloid-β peptide (Aβ). This satisfies the <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">'Amyloid Cascade Hypothesis'</a> as well as theories that see plaques as <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">neuroprotective</a>, and soluble Aβ as the real threat. MMP-9 is expressed at low basal levels in microglia and may keep plaque size in dynamic equilibrium <a href="http://www.ncbi.nlm.nih.gov/pubmed/16787929" target="_blank">(Yan et al. 2006)</a>. Over producing it in (inactive) GEM could greatly improve both soluble and insoluble Aβ clearance. MMP-9 must be delivered in GEM and expressed only in the vicinity of plaques, as otherwise it could cause damage to brain tissue if, for example, injected into the brain.
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This is a hormone containing 28 amino acid residues. It is a powerful anti-inflammatory neuropeptide. It exhibits neuroprotection in AD model systems <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC40251/" target="_blank">(Goze et al. 1996)</a>, able to save neurons exposed to excessive defective Aβ (70% success rate <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC40251/" target="_blank">(Goze et al. 1996)</a> by limiting neuroinflammation). VIP de-activates microglia <a href="http://www.ncbi.nlm.nih.gov/pubmed/12923064" target="_blank">(Delgado and Ganea 2003)</a>, inhibiting the production of inflammatory chemokines from microglia and reduces chemotaxis. VIP must be delivered in GEM and expressed only in the vicinity of plaques, as otherwise it could inactivate too much of the brain immune if, for example, injected into the brain. If constitutively expressed, it would stop GEM migrating to plaques. 
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While there is no change in BDNF levels in AD patients <a href="http://www.ncbi.nlm.nih.gov/pubmed/19363274" target="_blank">(O’Bryant et al. 2009)</a>, oxidative stress in older brains can increase levels of proteins which cause cell cycle re-entry and cell death, leading to AD <a href="http://www.ncbi.nlm.nih.gov/pubmed/20436277" target="_blank">(Frade & Lopez-Sanchez 2010)</a>. BDNF can combat these effects. BDNF is a trophic factor, able to help sustain dying brain cells. It can alter connectivity, synapse strength and neurogenesis and so must be delivered in GEM and expressed only in the vicinity of plaques, as otherwise it could more wildly change neuronal functions and circuits.
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This is a small non-inflammatory chemokine that induces chemotaxis [internal link to chemotaxis page] in macrophages. Microglia originate from a macrophage lineage. It elicits its effect through cell surface chemokine receptor CXCR3. Plaques already attract microglia, but this is partly due to local microglial activation. De-activated GEM will not produce many chemokines, so in order attract more GEM (as well as native microglia) to the plaque site, in order to speed up Aβ clearance.
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Revision as of 11:46, 6 September 2013

CIRCUIT OVERVIEW

IGEM: Intelligently Genetically Engineered Microglia

In order to direct microglia activity to senile plaques [internal link to neuropathology], we needed to find a way to detect plaques. Several possible routes were explored; initial focus was on a plaque binding protein, such as the B10 antibody (Haupt et al., 2011 [http://www.ncbi.nlm.nih.gov/pubmed/21059358]). 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 [http://www.ncbi.nlm.nih.gov/pubmed/10863548]); microglia are naturally attracted to plaques, and upon reaching plaques, a standard immune response follows, which 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 (cyc100 [http://parts.igem.org/Part:BBa_K105027]) already in the registry. Although from yeast, this 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 [http://www.ncbi.nlm.nih.gov/pubmed/17486122]). NF-κB is a transcription factor which translocates to the nucleus under oxidative stress (Shi et al., 2003 [http://www.ncbi.nlm.nih.gov/pubmed/12730877]), and binds to the sequence GGGAATTT (Park et al., 2009 [http://www.ncbi.nlm.nih.gov/pubmed/19435890]). 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 [link to experiments, linker construction] - short overlapping primers were ordered, and allowed to anneal, and then ligated together.

EXPERIMENTS AND RESULTS

Oxidative stress via free radical production increases with proximity to senile plaques (Colton et al., 2000). The microglia immune response around plaques also increases oxidative stress. Therefore, we have designed a promoter which will initiate transcription in response to oxidative stress to ensure the production of key proteins only in the plaques’ locales. This promoter is an improvement of a yeast minimal promoter (cyc100) already in the registry. 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.

MMP-9, also known as gelatinase B, is most commonly known for its role in breaking down the extracellular matrix. Naturally it is secreted in its inactive form and must be cleaved by other proteases, but our GEM are are meant to produce just the active form. It has been shown by Yan et al. that MMP-9 is the only known endogenous protease that degrades both fibrillar and soluble forms of amyloid-β peptide (Aβ). This satisfies the 'Amyloid Cascade Hypothesis' as well as theories that see plaques as neuroprotective, and soluble Aβ as the real threat. MMP-9 is expressed at low basal levels in microglia and may keep plaque size in dynamic equilibrium (Yan et al. 2006). Over producing it in (inactive) GEM could greatly improve both soluble and insoluble Aβ clearance. MMP-9 must be delivered in GEM and expressed only in the vicinity of plaques, as otherwise it could cause damage to brain tissue if, for example, injected into the brain.

This is a hormone containing 28 amino acid residues. It is a powerful anti-inflammatory neuropeptide. It exhibits neuroprotection in AD model systems (Goze et al. 1996), able to save neurons exposed to excessive defective Aβ (70% success rate (Goze et al. 1996) by limiting neuroinflammation). VIP de-activates microglia (Delgado and Ganea 2003), inhibiting the production of inflammatory chemokines from microglia and reduces chemotaxis. VIP must be delivered in GEM and expressed only in the vicinity of plaques, as otherwise it could inactivate too much of the brain immune if, for example, injected into the brain. If constitutively expressed, it would stop GEM migrating to plaques.

While there is no change in BDNF levels in AD patients (O’Bryant et al. 2009), oxidative stress in older brains can increase levels of proteins which cause cell cycle re-entry and cell death, leading to AD (Frade & Lopez-Sanchez 2010). BDNF can combat these effects. BDNF is a trophic factor, able to help sustain dying brain cells. It can alter connectivity, synapse strength and neurogenesis and so must be delivered in GEM and expressed only in the vicinity of plaques, as otherwise it could more wildly change neuronal functions and circuits.

This is a small non-inflammatory chemokine that induces chemotaxis [internal link to chemotaxis page] in macrophages. Microglia originate from a macrophage lineage. It elicits its effect through cell surface chemokine receptor CXCR3. Plaques already attract microglia, but this is partly due to local microglial activation. De-activated GEM will not produce many chemokines, so in order attract more GEM (as well as native microglia) to the plaque site, in order to speed up Aβ clearance.