Team:MIT/Treatment

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<li><a href="#top">Top</a></li>
 
<li><a href="#description">Description</a></li>
<li><a href="#description">Description</a></li>
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<li><a href="#outcome">Outcome</a></li>
 
<li><a href="#experiments">Experiments</a></li>
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<li><a href="#1">BACE1</a></li>
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<li><a href="#2">BACE2</a></li>
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<h1>Native Detector Module <a name="top" /></h1><br />
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<h3 align= "center" style="font-size:30px; color:teal">TREATMENT MODULE <a name="top" ></a></h3>
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<div style="float:left;clear:none"><img src="https://static.igem.org/mediawiki/2014/a/a7/MIT_protein_receptor.jpg"></div>
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<p style="font-size:12px" align=center><i><b>SUBGROUP MEMBERS: James T Anderson, Alex Laffell</b></i></p>
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<p style="font-size:12px" align=center><i>Attributions: James T Anderson</i></p>
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<font size=4><br />Down-regulating Aβ production and up-regulating Aβ degradation</font><br /><br />
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<option value="https://static.igem.org/mediawiki/2014/8/8b/MIT_treatment_animation_overview.png">Overview</option>
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by <a href="http://javascriptsource.com">The JavaScript Source</a></font>
by <a href="http://javascriptsource.com">The JavaScript Source</a></font>
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<center>Each of our three planned experiments for determining our effectiveness at re-regulating BACE1/BACE2 expression relied on successful transfection of our vectors into HEK293. Unfortunately, continual struggles with low transfection efficiency precluded us from performing all but our FRET experiment. From this experiment we dected less Bace1 activity in the four samples transfected with each of our four miRNA's.<br /></center>
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<h2>Outcome</h2><a name="outcome" ></a>
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<h2>Description</h2><a name="description"></a>
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The Treatment module is actuated upon release of transcription factor rtTA, initiating a two-part response. The first of these two responses is transcriptional up-regulation of an exogenously delivered vector expressing the Bace2 protease. Bace2 recognizes cleavage sites within Aß and so is thought to be a potentially effective therapeutic for degrading plaques. <br>
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<br>
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The second response activated by the release of rtTA is transcriptional expression of a miRNA targeted specifically to BACE1. We constructed four independent miRNA-generating vectors, each targeted to a different region of the BACE1 mRNA sequence. The benefit of having multiple unique miRNA’s is the ability to test the relative efficacy of each at down-regulating BACE1 translational expression. Predicting an effective miRNA depends on a variety of factors ranging from the sequence of bases that flank the guide region, to the degree of complementarity between the miRNA and its target, to the region of the target that the miRNA binds. <br>
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Vivamus maximus lectus diam, quis luctus tortor blandit sed. Pellentesque ullamcorper urna ut elit gravida, eget pretium orci maximus. Nam dignissim diam tristique placerat aliquet. Nullam nulla nulla, porttitor nec lorem quis, scelerisque rutrum odio. Suspendisse potenti. Vestibulum vel metus metus. Nam eu tempus mauris. Sed et mattis ex. Curabitur eu purus fringilla mi molestie fermentum sit amet non arcu. Aliquam erat volutpat.<br />
 
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<br><a href="#top">return to top</a>
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<h2>Experiments</h2><a name="experiments" ></a>
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<img src="https://static.igem.org/mediawiki/2014/1/17/MIT_results.jpg"><br><br><a href="#top">return to top</a>
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<a name="1"></a><h3>Transfection for BACE1 Experiments</h3></br>
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To test our miRNA’s effectiveness at down-regulating BACE1 expression we transfected separate HEK293 cultures with either (1) BACE1 under constitutive expression by the Hef1a promoter or (2) with BACE1 under constitutive expression by the Hef1a promoter and with a miRNA-generating vector inducibly regulated by the TRE promoter. We performed these transfections in duplicate—once with native BACE1, and then once with eYFP-tagged BACE1. There were two variants of this eYFP-tagged BACE1 construct—one with eYFP linked to the N-terminus of Bace1 and another with eYFP on the C-terminus. We created these two variants after considering the possibility that linking eYFP to either terminus of Bace1 might interfere with the peptide’s native structure or function. <br /> <br />
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After transfection and Doxycycline-induction of our miRNA-generating vectors, we used flow cytometry to verify that the miRNA-generating vectors were being expressed. Proper splicing of the mature miRNA out of the expression vector leaves an intact mKate coding sequence in the vector, so emission of red fluorescence from our transfected HEK293 indicates proper miRNA processing. <br /> <br />
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<table>
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<table>
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<tr><td width="100%">
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<table><tr>
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<td width="25%" align=center><p align=left><b>miRNA1</b><br></p>
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<a href="https://static.igem.org/mediawiki/2014/c/c7/MiRNA1.png">
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<img width="95%" src="https://static.igem.org/mediawiki/2014/c/c7/MiRNA1.png"></a></td>
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<td width="25%" align=center><p align=left><b>miRNA2</b><br></p>
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<a href="https://static.igem.org/mediawiki/2014/f/fa/MiRNA2.png">
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<img width="95%" src="https://static.igem.org/mediawiki/2014/f/fa/MiRNA2.png"></a></td>
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<td width="25%" align=center><p align=left><b>miRNA3</b><br></p>
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<a href="https://static.igem.org/mediawiki/2014/0/0d/MiRNA3.png">
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<img width="95%" src="https://static.igem.org/mediawiki/2014/0/0d/MiRNA3.png"></a></td>
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<td width="25%" align=center><p align=left><b>miRNA4</b><br></p>
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<a href="https://static.igem.org/mediawiki/2014/8/82/MiRNA4.png">
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<img width="95%" src="https://static.igem.org/mediawiki/2014/8/82/MiRNA4.png"></a></td>
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</tr>
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</table>
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<tr><td width="100%">
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<p> <b>Fig. 1. Determining whether miRNA's 1-4 are successfully processed.</b><i> The data represent transfection with Hef1a:BACE1, Hef1a:tagBFP, and each of the listed miRNA's. Expression of each miRNA was induced by treatment with 1uM Doxycycline. </i></p></td>
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</table><br><a href="#top">return to top</a>
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<a name="2"></a><h3>Transfection for BACE2 Experiments</h3></br>
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The one important difference between the BACE1 and BACE2 experiments was only that the BACE2 experiment required a simpler initial transfection. We transfected two cultures of HEK293 with either (1) a vector inducibly expressing BACE2 under control of the TRE promoter or (2) with a vector inducibly expressing BACE2 under control of a TRE promoter and with a vector constitutively expressing the rtTA transcription factor. So by comparing the amount of BACE2 expression in the induced versus un-induced transfection samples, we could determine our effectiveness at up-regulating BACE2 expression. <br /> <br />
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<h2>Experiments</h2><a name="experiments" ></a>
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Following confirmation of successful transfection via flow cytometry, we planned three experiments to assay the change in BACE1/2 expression. <br /> <br />
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<img src="https://static.igem.org/mediawiki/2014/1/17/MIT_results.jpg"><br>
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<b>First Experiment: FRET Assay</b><br /> <br />The first experiment was based on Anaspec’s SensoLyte 520 β-Secretase Assay Kit. This commercial kit uses a FRET-based technique wherein biological samples are mixed with a proprietary solution containing a BACE1/2 mimic substrate. One terminal of this mimic substrate is conjugated to a fluorophore and the other terminal is conjugated to a fluorescence quencher. So proteolytic Bace1/2 activity causes cleavage of the proprietary substrate, which increases fluorescence due to separation of the fluorophore from the quencher. Relative Bace1 activity in biological samples can be assayed by measuring the intensity of the fluorophore emission and comparing to a standard curve. <br /> <br />
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<img src="https://static.igem.org/mediawiki/2014/4/49/Bace1_anaspec.png" width=600px><br><br>
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Suspendisse luctus lobortis feugiat. Aenean vel velit rhoncus, molestie tellus non, egestas nulla. Interdum et malesuada fames ac ante ipsum primis in faucibus. Nam eleifend tortor eget tincidunt semper. Suspendisse porttitor mollis ullamcorper. Maecenas commodo ante nec diam rhoncus tristique. Ut varius leo a libero cursus posuere. Duis consectetur metus tincidunt mauris dictum venenatis. Maecenas sit amet urna elementum, tristique justo sed, eleifend ex. Fusce nec volutpat ligula, id venenatis ligula. Fusce in hendrerit tellus. Nam eu rutrum orci. Morbi ac augue vitae sapien varius posuere. Suspendisse sit amet odio est. Pellentesque rutrum, purus a faucibus dictum, magna libero iaculis odio, in dapibus nisl lacus sed tellus. Integer faucibus risus dolor, in blandit est interdum ut.
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<b>Fig.2. Determining miRNA efficacy in down-regulating BACE1 expression.</b><i> 30,000,000 cells from each sample were analyzed. RFU represents "relative fluorescence units", a proxy for determining Bace1 protease activity. The shown fluorescence values represent RFU after background fluorescence (as determinined by substrate-only control samples) was subtracted from raw output. </i><br/><br/>
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<b>Second Experiment: Detecting Fluorescence from YFP-tagged BACE1/2</b><br /> <br />The second planned experiment involved the previously mentioned eYFP-tagged Bace1/2 construct. Comparison of yellow fluorescence in miRNA-transfected versus miRNA-untransfected HEK293 would offer evidence as to whether BACE1/2 expression increases or decreases under the regulation of our miRNA’s. Furthermore, microscopy would allow us to visualize where Bace1/2 localizes in the cell. <br /> <br />
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<b>Third Experiment: RT-PCR</b><br /><br />
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Morbi aliquet lacus eros, condimentum vulputate lectus blandit vel. Morbi ac nibh a elit placerat convallis. Curabitur finibus nibh at diam aliquet, vel consequat turpis ullamcorper. Proin suscipit malesuada quam. In hac habitasse platea dictumst. Morbi posuere hendrerit ipsum, nec porta nisl sollicitudin vitae. In justo urna, hendrerit quis quam in, tempus ultricies velit. Fusce hendrerit, diam nec sodales dignissim, tortor nulla cursus massa, sed pharetra lacus magna at sapien. Praesent euismod sodales felis ut efficitur. Praesent lacinia, felis non efficitur condimentum, arcu dui mollis orci, non placerat libero ipsum eu leo. Ut cursus imperdiet nisi at aliquam. Sed leo tortor, blandit sed mattis sit amet, semper a erat. Donec consectetur velit id arcu viverra, a faucibus neque efficitur. Vestibulum ullamcorper elit arcu, id elementum nisi dignissim in. Etiam gravida nibh at neque elementum, id semper nisi pharetra. Integer quis ante non elit porta efficitur.
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Our third intended experiment was RT-PCR. We planned to isolate bulk RNA from the transfected HEK293, perform cDNA synthesis on the isolated RNA, and then use quantitative RT-PCR to determine the relative amount of Bace1/2 mRNA in miRNA-transfected versus miRNA-untransfected HEK293 to determine the efficacy of our miRNA’s in targeting BACE1/2 mRNA for degradation. <br /> <br />
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<h2>Parts</h2><a name="parts" ></a>
<h2>Parts</h2><a name="parts" ></a>
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<a href="https://2014.igem.org/Team:MIT/Parts">full parts list available here</a><br>
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• TRE: miRNA1 <br />
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• TRE: miRNA2 <br />
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• TRE: miRNA3 <br />
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• TRE: miRNA4 <br />
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• Hef1a: BACE1 <br />
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• Hef1a: eYFP-BACE1 <br />
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• Hef1a: BACE1-eYFP <br />
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• TRE: BACE2 <br />
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Etiam venenatis rutrum lorem, dignissim dignissim odio egestas in. Duis tempor ultricies porttitor. In vitae hendrerit est. Sed id dolor nec ante maximus vestibulum nec vel velit. Curabitur pellentesque varius dui sed sagittis. Praesent vitae enim id arcu dictum imperdiet in a libero. Morbi sit amet risus quis lectus vestibulum hendrerit. Etiam non consectetur justo, quis tincidunt ex. Nam varius arcu at quam blandit elementum. Nam sit amet odio a ante tristique molestie nec a turpis. Phasellus mi lorem, venenatis feugiat dignissim quis, gravida vitae nibh. Aliquam sodales ex enim, fringilla finibus ipsum tincidunt in. Donec felis tortor, auctor sit amet luctus non, mattis vitae dui.
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• TRE: eYFP-BACE2 <br />
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• TRE: BACE2-eYFP <br />
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</td>
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Latest revision as of 03:59, 18 October 2014

 


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TREATMENT MODULE

SUBGROUP MEMBERS: James T Anderson, Alex Laffell

Attributions: James T Anderson


Down-regulating Aβ production and up-regulating Aβ degradation



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Each of our three planned experiments for determining our effectiveness at re-regulating BACE1/BACE2 expression relied on successful transfection of our vectors into HEK293. Unfortunately, continual struggles with low transfection efficiency precluded us from performing all but our FRET experiment. From this experiment we dected less Bace1 activity in the four samples transfected with each of our four miRNA's.

Description

The Treatment module is actuated upon release of transcription factor rtTA, initiating a two-part response. The first of these two responses is transcriptional up-regulation of an exogenously delivered vector expressing the Bace2 protease. Bace2 recognizes cleavage sites within Aß and so is thought to be a potentially effective therapeutic for degrading plaques.

The second response activated by the release of rtTA is transcriptional expression of a miRNA targeted specifically to BACE1. We constructed four independent miRNA-generating vectors, each targeted to a different region of the BACE1 mRNA sequence. The benefit of having multiple unique miRNA’s is the ability to test the relative efficacy of each at down-regulating BACE1 translational expression. Predicting an effective miRNA depends on a variety of factors ranging from the sequence of bases that flank the guide region, to the degree of complementarity between the miRNA and its target, to the region of the target that the miRNA binds.

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Experiments



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Transfection for BACE1 Experiments


To test our miRNA’s effectiveness at down-regulating BACE1 expression we transfected separate HEK293 cultures with either (1) BACE1 under constitutive expression by the Hef1a promoter or (2) with BACE1 under constitutive expression by the Hef1a promoter and with a miRNA-generating vector inducibly regulated by the TRE promoter. We performed these transfections in duplicate—once with native BACE1, and then once with eYFP-tagged BACE1. There were two variants of this eYFP-tagged BACE1 construct—one with eYFP linked to the N-terminus of Bace1 and another with eYFP on the C-terminus. We created these two variants after considering the possibility that linking eYFP to either terminus of Bace1 might interfere with the peptide’s native structure or function.

After transfection and Doxycycline-induction of our miRNA-generating vectors, we used flow cytometry to verify that the miRNA-generating vectors were being expressed. Proper splicing of the mature miRNA out of the expression vector leaves an intact mKate coding sequence in the vector, so emission of red fluorescence from our transfected HEK293 indicates proper miRNA processing.

miRNA1

miRNA2

miRNA3

miRNA4

Fig. 1. Determining whether miRNA's 1-4 are successfully processed. The data represent transfection with Hef1a:BACE1, Hef1a:tagBFP, and each of the listed miRNA's. Expression of each miRNA was induced by treatment with 1uM Doxycycline.


return to top

Transfection for BACE2 Experiments


The one important difference between the BACE1 and BACE2 experiments was only that the BACE2 experiment required a simpler initial transfection. We transfected two cultures of HEK293 with either (1) a vector inducibly expressing BACE2 under control of the TRE promoter or (2) with a vector inducibly expressing BACE2 under control of a TRE promoter and with a vector constitutively expressing the rtTA transcription factor. So by comparing the amount of BACE2 expression in the induced versus un-induced transfection samples, we could determine our effectiveness at up-regulating BACE2 expression.

Following confirmation of successful transfection via flow cytometry, we planned three experiments to assay the change in BACE1/2 expression.

First Experiment: FRET Assay

The first experiment was based on Anaspec’s SensoLyte 520 β-Secretase Assay Kit. This commercial kit uses a FRET-based technique wherein biological samples are mixed with a proprietary solution containing a BACE1/2 mimic substrate. One terminal of this mimic substrate is conjugated to a fluorophore and the other terminal is conjugated to a fluorescence quencher. So proteolytic Bace1/2 activity causes cleavage of the proprietary substrate, which increases fluorescence due to separation of the fluorophore from the quencher. Relative Bace1 activity in biological samples can be assayed by measuring the intensity of the fluorophore emission and comparing to a standard curve.



Fig.2. Determining miRNA efficacy in down-regulating BACE1 expression. 30,000,000 cells from each sample were analyzed. RFU represents "relative fluorescence units", a proxy for determining Bace1 protease activity. The shown fluorescence values represent RFU after background fluorescence (as determinined by substrate-only control samples) was subtracted from raw output.

Second Experiment: Detecting Fluorescence from YFP-tagged BACE1/2

The second planned experiment involved the previously mentioned eYFP-tagged Bace1/2 construct. Comparison of yellow fluorescence in miRNA-transfected versus miRNA-untransfected HEK293 would offer evidence as to whether BACE1/2 expression increases or decreases under the regulation of our miRNA’s. Furthermore, microscopy would allow us to visualize where Bace1/2 localizes in the cell.

Third Experiment: RT-PCR

Our third intended experiment was RT-PCR. We planned to isolate bulk RNA from the transfected HEK293, perform cDNA synthesis on the isolated RNA, and then use quantitative RT-PCR to determine the relative amount of Bace1/2 mRNA in miRNA-transfected versus miRNA-untransfected HEK293 to determine the efficacy of our miRNA’s in targeting BACE1/2 mRNA for degradation.


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Parts

full parts list available here
• TRE: miRNA1
• TRE: miRNA2
• TRE: miRNA3
• TRE: miRNA4
• Hef1a: BACE1
• Hef1a: eYFP-BACE1
• Hef1a: BACE1-eYFP
• TRE: BACE2
• TRE: eYFP-BACE2
• TRE: BACE2-eYFP

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