Functionally Complete Excision of Conditional Alleles in the Mouse Suprachiasmatic Nucleus by Vgat-ires-Cre

Abstract

Mice with targeted gene disruption have provided important information about the molecular mechanisms of circadian clock function. A full understanding of the roles of circadian-relevant genes requires manipulation of their expression in a tissue-specific manner, ideally including manipulation with high efficiency within the suprachiasmatic nuclei (SCN). To date, conditional manipulation of genes within the SCN has been difficult. In a previously developed mouse line, Cre recombinase was inserted into the vesicular GABA transporter (Vgat) locus. Since virtually all SCN neurons are GABAergic, this Vgat-Cre line seemed likely to have high efficiency at disrupting conditional alleles in SCN. To test this premise, the efficacy of Vgat-Cre in excising conditional (fl, for flanked by LoxP) alleles in the SCN was examined. Vgat-Cre-mediated excision of conditional alleles of Clock or Bmal1 led to loss of immunostaining for products of the targeted genes in the SCN. Vgat-Cre; Clock; Npas2 mice and Vgat-Cre; Bmal1 mice became arrhythmic immediately upon exposure to constant darkness, as expected based on the phenotype of mice in which these genes are disrupted throughout the body. The phenotype of mice with other combinations of Vgat-Cre, conditional Clock, and mutant Npas2 alleles also resembled the corresponding whole-body knockout mice. These data indicate that the Vgat-Cre line is useful for Cre-mediated recombination within the SCN, making it useful for Cre-enabled technologies including gene disruption, gene replacement, and opto- and chemogenetic manipulation of the SCN circadian clock.

Desynchrony between brain and peripheral clocks caused by CK1δ/ε disruption in GABA neurons does not lead to adverse metabolic outcomes

“…Discordant Mice Have a Slow but Robust Central Circadian Clock. The GABAergic nature of SCN neurons (44) allowed the use of the Vgat-Cre + driver to target conditional alleles of CK1δ and CK1« (12) in the SCN and other GABAergic brain areas (45,46), although not directly affecting circadian clocks in peripheral tissues. Deletion of different combinations of CK1δ/« alleles in GABAergic neurons significantly lengthened the circadian period of both behavioral (F 5,150 = 356.5, P < 0.0001; Fig.…”

Clocks and meals keep mice from being cool

“…The assessment of the role of food timing in the regulation of torpor might thus be confounded by the presence of circadian regulation. To study the isolated role of food timing in controlling the timing of torpor, we next disrupted endogenous circadian rhythmicity by disrupting the essential clock gene Bmal1 in GABAergic neurons including the SCN, while leaving clocks in peripheral tissues effectively wildtype (Vgat-Cre + Bmal1 fl/fl ; Weaver et al, 2018). Food was provided at 24 h (2.2 g) or 20 h (1.8 g) intervals (Fig.…”

“…Six female wild-type C57BL/6J mice were purchased from Jackson Laboratories. Vgat-Cre + CK1δ fl/fl ε fl/+ mice (5 male, 4 female) (van der Vinne et al, 2018), Vgat-Cre + Bmal1 fl/fl mice (5 male, 5 female) and No-Cre Bmal1 fl/fl mice (5 male, 7 female) (Weaver et al, 2018) were generated by crossing previously described genotypes (Etchegaray et al, 2009;Storch et al, 2007;Vong et al, 2011). Genotyping was performed using ear or tail biopsies by PCR as described previously (Etchegaray et al, 2009;Weaver et al, 2018).…”

Feeding Rhythms and the Circadian Regulation of Metabolism

“…Indeed, the SCN is necessary to drive behavioral and feeding rhythms. Early studies of SCN lesions (29) and recent SCN-enriched selective knockouts of clock genes (30,31) confirmed that without a functional central clock, animals in constant darkness are arrhythmic in their FIGURE 1 | The mammalian molecular clock. The complex of BMAL1 and CLOCK rhythmically binds E-box elements to activate the transcription of clock-controlled genes (CCG), including other parts of the core clock which form the negative arm of the feedback loop.…”