A lentiviral vector bearing a reverse intron demonstrates superior expression of both proteins and microRNAs

Abstract

While lentiviral expression vectors are widely used in many facets of molecular biology, due to their ability to stably express heterologous genes in both dividing and non-dividing cells, they suffer from the disadvantage that introns inserted into the vector genome are generally rapidly lost by splicing in packaging cell lines. The presence of an intron, if achievable, has the potential to facilitate the expression of transgene cDNAs, as splicing has been extensively shown to facilitate mRNA biogenesis and function. Moreover, if a stable intron could be introduced into a lentiviral vector, this could greatly facilitate the expression of microRNAs (miRNAs), and especially miRNA clusters, as the introduction of pri-miRNA stems into the exonic region of a lentiviral vector can strongly reduce both vector titer and the expression of any miRNA-linked indicator gene due to cleavage of the vector RNA genome by cellular Drosha. Here, we describe a novel lentiviral vector design in which transgenes and/or miRNAs are expressed using an antisense-orientated, inducible promoter driving an expression cassette bearing a functional intron. We demonstrate that this lentiviral vector, called pTREX, is able to express higher levels of both transgenes and pri-miRNA clusters when compared with a closely similar conventional lentiviral vector.

The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis

“…To this end, the miR-BHRF1-1, 1-2 and 1-3 pri-miRNAs were cloned into the antisense-oriented intron present in the Tet-inducible lentiviral miRNA expression vector pTREX, as this vector allows a consistently higher level of pri-miRNA expression when compared to conventional lentiviral vectors (Fig. 1B) (Poling et al, 2017). miR-BHRF1-1 and 1-3 are not well expressed from their natural pri-miRNA stem-loops, isolated from the EBV genome (Feederle et al, 2011a; Haar et al, 2015), so they were expressed from expression cassettes based on the human pri-miR-30 precursor, as previously described (Zeng et al, 2002).…”

“…All miRNA and amiRNA expression vectors were generated by cloning into the pTREX vector using the XhoI and EcoRI sites present within the intron upstream of the Thy1.1 protein (primers 1–3 in Supplementary Table 1) (Poling et al, 2017). The pTREX BHRF1-123 miRNA expression vector was previously described (Poling et al, 2017).…”

“…The pTREX BHRF1-123 miRNA expression vector was previously described (Poling et al, 2017). amiRNAs targeting the BHRF1 3′UTR, and a control FLuc amiRNA, were generated in the pri-miR-30 context, as previously described (Zeng et al, 2002), and then cloned into pTREX.…”

“…miR-BHRF1 levels were determined by stem-loop real-time PCR as previously described (Chen et al, 2005; Feederle et al, 2011b; Poling et al, 2017). Briefly, total RNA was harvested from LCLs using TRIzol (LifeTechnologies) extraction following the manufacturer’s protocol except that the last 70% ethanol wash was replaced with a re-precipitation.…”

“…*p<0.05, **p<0.01; one-sample t-test. (B) Schematic of the pTREX vector used to express the BHRF1 miRNAs (Poling et al, 2017). Pri-miRNAs, with the mature miRNAs shown in red, were cloned between the XhoI and EcoRI sites present inside the intron 5′ of Thy1.1.…”