Part:BBa_K1391129:Experience

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Applications of BBa_K1391129

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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