Investigation of the SCN 2.2 Cell Line as a Model of the Central Mammalian Circadian Pacemaker

129 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2001.The SCN 2.2 cell line, derived by immortalization of fetal rat suprachiasmatic nucleus (SCN) tissue, was investigated for characteristics of the SCN, which is the central mammalian circadian pacemaker. Extensive characterization of differentiated SCN 2.2 cultures revealed that this cell line expresses putative clock genes, as well as components of daytime, nighttime and crepuscular circadian regulatory pathways. The line also expresses several antigens that are highly expressed and/or relatively restricted within the hypothalamus to the SCN. Stable SCN 2.2 reporter cell lines were prepared that expressed luciferase activity driven by cAMP response element (SCN 2.2.cre-luc), mouse period 1 E-box (SCN 2.2.ebox-luc) and human vasoactive intestinal peptide (SCN2.2.vip-luc) promoters. Serumshocked stably transfected SCN 2.2 cell lines displayed near-24 hour rhythms. These rhythms were blocked by inhibition of cGMP-dependent protein kinase (PKG), a known element of several circadian regulatory pathways. As with the SCN, synchronized SCN 2.2 cultures expressed a gated response to glutamate stimulus in both induction of ser-133 phosphorylated cAMP response element binding protein (PCREB) and luciferase activity in SCN 2.2.cre-luc, SCN 2.2.ebox-luc and SCN 2.2. vip-luc cultures. Characteristics such as expression of circadian regulatory proteins and putative clock genes, rhythmic gene expression and gated response to stimulus suggest that the SCN 2.2 line is an appropriate model for the central mammalian pacemaker. Furthermore, results derived from the SCN 2.2 cell line experiments enabled a prediction of transcriptional mediators of light/glutamate phase-shifting signals that was verified by experiments in the SCN.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD