Long-term transgene expression in mouse neural progenitor cells modified with PhiC31 integrase

Authors:
Keravala A, Ormerod BK, Palmer TD, Calos MP
In:
Source: J Neurosci Methods
Publication Date: (2008)
Issue: 173(2): 299-305
Research Area:
Neurobiology
Stem Cells
Cells used in publication:
Neural stem cell (NSC), mouse
Species: mouse
Tissue Origin: brain
Platform:
Nucleofectorâ„¢ I/II/2b
Experiment
Nucleofection of mNPCs with plasmid coding for PhiC31 Inegrase along with either a plasmid containing AttB CMV and GFP or a plasmid containing AttB CMV and Luciferase to generate targeted clones.
Abstract
Stem cells can potentially be utilized in combined gene/cell therapies for neural diseases. We examined the ability of the non-viral varphiC31 integrase system to promote stable transgene expression in mouse neural progenitor cells (mNPCs). varphiC31 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 varphiC31 integrase system produces stable transgene expression in adult mNPCs and their progeny and may be useful in strategies for combating neurodegenerative disorders.