pLemiR Vector Details
Vector Element	Utility
CMV	RNA Polymerase II promoter
cPPT	Central Polypurine tract - helps translocation into the nucleus of non-dividing cells
WRE	Enhances the stability and translation of transcripts
TurboRFP	Marker to track inducible shRNAmir expression
Puror	Mammalian selectable marker
RRE	Rev Response element
5'LTR	Wildtype 5'long terminal repeat
SIN-LTR	3' Self-inactivating long terminal repeat
ZEOr	Bacterial selectable marker

Insert sequences in the miR-express miRNA clones contain not only the pre-miRNA sequence but also incorporate flanking genomic sequences on both the 5’ and 3’ ends of the pre-miRNA, the presumed primary miRNA sequence including regulatory elements (see below).

An example of a construct from the miR-express lentiviral library

A. The pri-miRNA sequence with pre-miRNA hairpin in bold represents the entire insert sequence. B. The pre-miR-101-2 stem-loop structure and resulting mature miRNA sequence (red) is shown in lower panel http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000739)

The miR-express™ microRNA is transcribed on the same transcript as the TurboRFP enabling visual tracking of microRNA expression

Tracking microRNA expression with TurboRFP

Lentivirus expressed native pri-miRNA precursors are processed into mature miRNA duplexes by the native RNAi machinery in transduced HEK293 cells. The microscopic images show that the miRNA expression is trackable via TurboRFP production. Viral stocks were produced using the TransLentiviral™ Packaging System modified for use with a CaPO4 transfection protocol.