| Types | DnaRegion
|
| Roles | plasmid
Plasmid
|
| Sequences | BBa_K1391136_sequence (Version 1)
|
Description
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.
Notes
There are a variety of factors to consider when designing a miRNA to effectively down-regulate a gene of interest. Among these are the region within the gene that the miRNA binds, the degree of complementarity between the miRNA and its target sequence, the bases flanking the mature miRNA sequence, and non-specific down-regulation of other undesirable targets. To optimize these parameters we referred to online tools such as Ohio State University???s miR-Synth tool.
Source
The miRNA sequence was assembled via Golden-Gate with two G-blocks. The miRNA sequence was then assembled with the promoter part pENTR TRE by Gateway.
