XMIRXpress Lentivector System

The XMIRXpress lentivectors are based on the same XMotif exosomal targeting RNA tag utilized in the XMIR/AXMIR synthetic oligos. There are a number of pre-made XMIRXpress miRNA expression constructs available, and SBI will design and build a custom XMIRXpress lentivector construct for any miRNA, anti-miRNA or siRNA of choice for the same list price as the pre-made constructs. The lentivectors all feature an EF1a-GFP-Puro selection cassette and a downstream H1 promoter expressing the XMIR + XMotif cassette. In additiona to pre-made XMIRXpress lentivectors, a cloning XMIRXpress lentivector (cat# XMIRXP-VECT) is available to fuse the XMotif to any miRNA, anti-miRNA, or siRNA of choice, which can then be used to generate stable cellular exosome "factories".

XMIRXpress XMIRXP-1 loading into exosomes

XMIR-29b lentivector expression construct was transfected into HEK293 cells cultured in DMEM media with SBI's exosome-depleted FBS media supplement in place of standard FBS to remove contaminating bovine exosomes. HEK293 exosomes were collected after 48 hours. The exosomal RNA was purified and converted into qPCR-compatible cDNA. Relative amounts of XMIR-29b packaged into exosomes from the XMIRXP-29b lentivector were quantitated by qPCR with miR-16 used as a reference exosome control signal. The delta-delta Ct calculation for XMIR-29b is shown in the bar graph.

XMIRXpress cloning and expression lentivector

SBI also provides a cloning and expression XMIRXpress lentivector plasmid (cat# XMIRXP-VECT) to allow you to clone any miRNA or anti-miRNA of your choice and fuse it to the XMotif sequence upon ligation. The XMIRXP-VECT plasmid is provided in a linearized form for rapid cloning. Simply design two DNA oligos for the top and bottom strand as detailed below, anneal the top and bottom oligos, and ligate directly into the XMIRXP-VECT plasmid. An example of cloning hsa-miR-29b (MI0000105) is shown below. First, look up miRNA of interest using miRBase (www.miRBase.org) and identify what miRNA hairpin sequence you wish to express as an XMIR. Convert the U residues to T residues and create the top and bottom strand oligos with restriction enzyme site overhangs as shown below. The XMIRXpress lentivectors can be used in transfection experiments to test the exosomal loading of the XMIR and can also be packaged into lentivirus to easily create stable XMIR producer cell lines constitutively secreting exosomes packed with XMIRs. To create stable cell lines stably expressing XMIRs that will be loaded into secreted exosomes, package the XMIRXpress lentivector plasmid into virus using SBI's lentivirus packaging systems. Visit Lentiviral packaging systems for product information on the kits to perform virus packaging.