title: Long-term transgene expression in mouse neural progenitor cells modified with phiC31 integrase.
abstract: Stem cells can potentially be utilized in combined gene/cell therapies for neural diseases. We examined the ability of the non-viral phiC31 integrase system to promote stable transgene expression in mouse neural progenitor cells (mNPCs). phiC31 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 phiC31 integrase system produces stable transgene expression in adult mNPCs and their progeny and may be useful in strategies for combating neurodegenerative disorders.
authors.affiliation: Department of Genetics, Stanford University School of Medicine, M-334, 300 Pasteur Drive, Stanford, CA 94305-5120, United States.
authors.forename: Annahita
authors.initials: A
authors.lastname: Keravala
authors.forename: Brandi K
authors.initials: BK
authors.lastname: Ormerod
authors.forename: Theo D
authors.initials: TD
authors.lastname: Palmer
authors.forename: Michele P
authors.initials: MP
authors.lastname: Calos
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006085:Graft Survival / Q000235:genetics; D019426:Integrases / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D018874:Spheroids, Cellular; D033581:Stem Cell Transplantation / Q000379:methods*; D013234:Stem Cells / Q000378:metabolism*; D013995:Time; D013997:Time Factors; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
chemical_list: D008156:Luciferases; D019426:Integrases