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Team:MIT/Treatment
From 2014.igem.org
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| - | <p> <b>Determining whether miRNA's 1-4 are successfully processed.</b><i> The data represent transfection with Hef1a:BACE1, Hef1a:tagBFP, and <b>(A)</b> TRE:miRNA1, <b>(B)</b> TRE:miRNA2, <b>(C)</b> TRE:miRNA3, <b>(D)</b> TRE:miRNA4. Expression of each miRNA was induced by treatment with 1uM Doxycycline. </i></p> | + | <p> <b>Determining whether miRNA's 1-4 are successfully processed.</b><i> The data represent transfection with Hef1a:BACE1, Hef1a:tagBFP, and <b>(A)</b> TRE:miRNA1, <b>(B)</b> TRE:miRNA2, <b>(C)</b> TRE:miRNA3, <b>(D)</b> TRE:miRNA4. Expression of each miRNA was induced by treatment with 1uM Doxycycline. </i></p></td> |
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Revision as of 23:41, 17 October 2014
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Treatment ModuleDown-regulating Aβ production and up-regulating Aβ degradation
DescriptionThe 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. OutcomeEach 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. |
