Long-term transgene expression in mouse neural progenitor cells modified with ϕC31 integrase

Abstract

Stem cells can potentially be utilized in combined gene/cell therapies for neural diseases. We examined the ability of the non-viral ϕC31 integrase system to promote stable transgene expression in mouse neural progenitor cells (mNPCs). ϕC31 integrase catalyzes the sequence-specific integration of attB-containing plasmids into pseudo attP sites in mammalian genomes, to produce long-term transgene expression. We achieved gene transfer by co-nucleofection of a plasmid carrying the luciferase marker gene and an attB site and a plasmid expressing integrase in mNPCs that had been generated in a neurosphere preparation. Luciferase expression was quantified in live cells for 8 weeks, revealing persistence of gene expression. Sequence-specific integration at a preferred pseudo attP site in the mouse genome was detected by using PCR. Furthermore, sustained transgene expression was demonstrated in genetically modified NPCs that were cultured in conditions that promoted either growth or differentiation into neurons and astrocytes. Our results demonstrate that the ϕC31 integrase system produces stable transgene expression in adult mNPCs and their progeny and may be useful in strategies for combating neurodegenerative disorders.

The transfection of multipotent neural precursor/stem cell transplant populations with magnetic nanoparticles

“…demyelination), it is undesirable to maintain expression of a particular factor for prolonged periods as repair is regulated by dynamic, temporally controlled molecular expression patterns [49]. However, stable gene expression in NPCs using non-viral methods of transfection can be achieved (for example, by using plasmids containing appropriate promoters and conferring neomycin resistance, coupled with G418 selection [36] or by using the FC31 integrase system [45]), of relevance to conditions where long-term gene transfer may be required (e.g. amyotrophic lateral sclerosis, Parkinson's disease).…”

A Profile of Native Integration Sites Used by φC31 Integrase in the Bovine Genome

“…This enzyme can also effectively integrate attB-containing transgenes directly into mammalian genomes at endogenous attPlike sequences named pseudo attP sites (Thyagarajan et al, 2001). Numerous studies have demonstrated that 4C31 integrase is a useful tool for both gene therapy (Olivares et al, 2002;Calos, 2006;Aneja et al, 2007;Keravala et al, 2008;Woodard et al, 2010) and generation of transgenic animals (Hollis et al, 2003;Allen and Weeks, 2005;Fish et al, 2007).…”

Site-Specific Recombination Using PhiC31 Integrase

“…In this study, mouse neural progenitor cells were transfected with a donor plasmid expressing luciferase and kanamycin resistance and an integrase-expressing plasmid (Keravala et al 2008 ) . After 8 weeks of culture in selective media, the cells that received the integrase plasmid had 39-fold higher levels of luciferase expression than the cells that did not receive integrase.…”

“…After 8 weeks of culture in selective media, the cells that received the integrase plasmid had 39-fold higher levels of luciferase expression than the cells that did not receive integrase. Selective culture with G418 resulted in 201 resistant neurospheres in cells that received the integrase plasmid, compared to fi ve resistant neurospheres in cells that did not (Keravala et al 2008 ) . Integration was con fi rmed via polymerase chain reaction to have occurred at the pseudo attP hotspot mpsL1, and cells containing integration events mediated by f C31 integrase were observed to retain both their proliferative and differentiation capacities.…”