Team:MIT/Treatment

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Description
Outcome
Experiments
Parts

Treatment Module

Down-regulating Aβ production and up-regulating Aβ degradation

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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.

Outcome

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 any of our intended experiments. Analysis of our transfected HEK293 cultures with flow cytometry revealed very low numbers of live cells, suggesting that something had gone awry in the transfection protocol, in the quality of our vector constructs, or in our FACS preparation process. In any case, the lack of usable HEK293 meant we had no samples on which to perform any of our three intended experiments. This is an issue we hope to resolve soon so that we can make meaningful conclusions about the success of the Treatment module.

Experiments

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.

A	B
C	D
E	F

Determining whether miRNA's 1-4 are successfully spliced and processed Samples were transfected with Hef1a:BACE1, Hef1a:tagBFP (as a transfection marker), and TRE:miRNA. Expression of each miRNA was induced by treatment with 1uM Doxycycline. The data represent transfection with Hef1a:BACE1 and: (A) Hef1a:tagBFP, (B) Hef1a:tagBFP and Hef1a:mKate (C) Hef1a:tagBFP and TRE:miRNA1, (D) Hef1a:tagBFP and TRE:miRNA2, (E) Hef1a:tagBFP and TRE:miRNA3, (F) Hef1a:tagBFP and TRE:miRNA4

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 expression by miRNA’s.

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 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 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.

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

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

Parts

• TRE: miRNA1
• TRE: miRNA2
• TRE: miRNA3
• TRE: miRNA4
• Hef1a: BACE1
• Hef1a: eYFP-BACE1
• Hef1a: BACE1-eYFP
• TRE: BACE2
• TRE: eYFP-BACE2
• TRE: BACE2-eYFP

Retrieved from "http://2014.igem.org/Team:MIT/Treatment"

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-<p> <b>Determining membrane localization was complicated by bleedthrough from mKate into the yellow fluorescence channel.</b> Samples were stained with Alexa fluor 488 (yellow) conjugated anti-IgM antibodies and evaluated using flow cytometry. Red fluorescence refers to the amount of mKate (transfection marker) in the cells whereas yellow fluorescence measures the amount of anti-IgM binding (indicating the presence of B-cell receptors on the cell surface). (A) Untransfected, unstained HEK293; (B) HEK293 transfected with dummy DNA, stained; (C) HEK293 transfected with mKate, stained; (D) HEK293 transfected with mKate and BCR components, stained.</p>
+<p> <b>Determining whether miRNA's 1-4 are successfully spliced and processed</b> Samples were transfected with Hef1a:BACE1, Hef1a:tagBFP (as a transfection marker), and TRE:miRNA. Expression of each miRNA was induced by treatment with 1uM Doxycycline. The data represent transfection with Hef1a:BACE1 and: (A) Hef1a:tagBFP, (B) Hef1a:tagBFP and Hef1a:mKate (C) Hef1a:tagBFP and TRE:miRNA1, (D) Hef1a:tagBFP and TRE:miRNA2, (E) Hef1a:tagBFP and TRE:miRNA3, (F) Hef1a:tagBFP and TRE:miRNA4</p>
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