Analysis of circadian mechanisms in the suprachiasmatic nucleus by transgenesis and biolistic transfection.
Analysis of the cellular and molecular mechanisms that underlie the circadian pacemaker of the suprachiasmatic nuclei (SCN) requires in vitro preparations amenable to genetic manipulation that can provide dynamic measures of circadian activity in real time over multiple circadian cycles. This article focuses on the value of the SCN organotypic slice for such studies. Specifically, it describes the use of tissues from genetically modified mice in which the circadian promoter of the mPer1 gene is used to drive the expression of either firefly luciferase or destabilized green fluorescent protein optical reporters. Furthermore, we describe a procedure for biolistic (particle-mediated) transfection of SCN organotypic slices with fluorescent reporters that can be used to explore the cis-acting elements and trans-acting factors that control circadian patterning, and also the interactions between subpopulations of neuronal oscillators within the SCN assemblage.
Hastings
Michael H
MH
MRC Laboratory of Molecular Biology, Division of Neurobiology, Cambridge CB2 2QH, United Kingdom.
Reddy
Akhilesh B
AB
McMahon
Douglas G
DG
Maywood
Elisabeth S
ES
D000818:Animals; D019470:Biolistics / Q000379:methods*; D018797:Cell Cycle Proteins / Q000502:physiology; D002940:Circadian Rhythm / Q000502:physiology*; D018014:Gene Transfer Techniques; D051379:Mice; D009474:Neurons / Q000378:metabolism; D009687:Nuclear Proteins / Q000502:physiology; D009924:Organ Culture Techniques / Q000379:methods; D056950:Period Circadian Proteins; D013050:Spectrometry, Fluorescence; D013493:Suprachiasmatic Nucleus / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology; D014162:Transfection / Q000379:methods*
D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; C539023:Per1 protein, mouse; C513312:Per2 protein, mouse; D056950:Period Circadian Proteins; D014157:Transcription Factors