Novel strategy to study gene expression and function in developing cerebellar granule cells.
The advent of techniques for global analyses of cell biology, such as genomics and proteomics, opens the way to rapid progress in understanding the molecular control of developing tissues. However, such studies in the CNS are hindered by the complexity of this tissue. In particular, few approaches allow cells to be isolated that are enriched for specific stages of their maturation. We describe a new strategy to study gene expression and function in cerebellar granule cells. In these experiments, we have used square pulse electroporation to introduce fluorescent dye or DNA constructs into immature granule cell precursors in situ. This method only labels granule cell precursors in the superficial part of the external granule layer. Combining this labelling with fluorescent activated cell sorting (FACS) allows the transfected cells to be isolated at any time during their subsequent development, thus providing a means of analysing granule cells as they undergo maturation. This transfection method can be used to study events in the normal maturation of granule cells or the effects of introduced transgenes. Such studies can be carried out on cells purified from primary cultures or cells in situ using cerebellar slice cultures. Our strategy provides a new route to detailed analysis of the role of genes in controlling many aspects of granule cell biology. These approaches will allow recent global analyses to be more readily applied to subpopulations of cells in complex tissues.
Yang
Zeng-jie
ZJ
Children's Brain Tumour Research Centre, Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.
Appleby
Vanessa J
VJ
Coyle
Beth
B
Chan
Wai-In
WI
Tahmaseb
Mohammad
M
Wigmore
Peter M
PM
Scotting
Paul J
PJ
D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation / Q000235:genetics*; D002470:Cell Survival / Q000235:genetics; D002525:Cerebellar Cortex / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000494:pharmacology; D018274:Electroporation / Q000295:instrumentation / Q000379:methods; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D049452:Green Fluorescent Proteins; D066298:In Vitro Techniques; D008164:Luminescent Proteins; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014162:Transfection / Q000295:instrumentation / Q000379:methods*
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D004247:DNA