A Cre-lox approach for transient transgene expression in neural precursor cells and long-term tracking of their progeny in vitro and in vivo.
BACKGROUND
Neural precursor cells (NPCs) can be isolated from various regions of the postnatal central nervous system (CNS). Manipulation of gene expression in these cells offers a promising strategy to manipulate their fate both in vitro and in vivo. In this study, we developed a technique that allows the transient manipulation of single/multiple gene expression in NPCs in vitro, and the long-term tracking of their progeny both in vitro and in vivo.


RESULTS
In order to combine the advantages of transient transfection with the long-term tracking of the transfected cells progeny, we developed a new approach based on the cre-lox technology. We first established a fast and reliable protocol to isolate and culture NPCs as monolayer, from the spinal cord of neonatal transgenic Rosa26-YFP cre-reporter mice. These cells could be reliably transfected with single/multiple plasmids by nucleofection. Nucleofection with mono- or bicistronic plasmids containing the Cre recombinase gene resulted in efficient recombination and the long-term expression of the YFP-reporter gene. The transient cre-expression was non-toxic for the transfected cells and did not alter their intrinsic properties. Finally, we demonstrated that cre-transfected cells could be transplanted into the adult brain, where they maintained YFP expression permitting long-term tracking of their migration and differentiation.


CONCLUSIONS
This approach allows single/multiple genes to be manipulated in NPCs, while at the same time allowing long-term tracking of the transfected cells progeny to be analyzed both in vitro and in vivo.
Geoffroy
Cédric G
CG
Department of Clinical Neurosciences, Cambridge centre for Brain Repair, University of Cambridge, Cambridge, UK. cg355@cam.ac.uk <cg355@cam.ac.uk>
Raineteau
Olivier
O
D000818:Animals; D002478:Cells, Cultured; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D019426:Integrases / Q000235:genetics*; D051379:Mice; D009474:Neurons / Q000166:cytology* / Q000637:transplantation; D010957:Plasmids; D051127:Scavenger Receptors, Class E / Q000235:genetics*; D013116:Spinal Cord / Q000166:cytology; D033581:Stem Cell Transplantation; D019076:Transgenes
C495138:Olr1 protein, mouse; D051127:Scavenger Receptors, Class E; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases