Part:BBa_K1391136

pEXPR TRE:miRNAg2

To test the effectiveness of this miRNA, we co-transfected pEXPR TRE:miRNA with constitutive pEXPR Hef1a:BACE1. We then induced expression of pEXPR TRE:miRNA by Doxycycline treatment and compared BACE1 expression in the miRNA-transfected HEK293 versus HEK293 that received only Hef1a:BACE1 and no TRE:miRNA. BACE1 activity was quantified using the SensoLyte 520 β-Secretase Assay Kit (Anaspec). BACE1 activity decreased by about 50% when TRE:miRNA was co-transfected.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 3088
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 3088
Illegal NheI site found at 2124
Illegal NheI site found at 2390
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 3088
Illegal BamHI site found at 2554
Illegal XhoI site found at 2984
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 3088
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 3088
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 3610
Illegal SapI.rc site found at 2001
Illegal SapI.rc site found at 2602

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.