Limitations of the Avp-IRES2-Cre (JAX #023530) and Vip-IRES-Cre (JAX #010908) Models for Chronobiological Investigations

Abstract

The principal circadian pacemaker in mammals, the suprachiasmatic nucleus (SCN), expresses a number of neuropeptides that facilitate intercellular synchrony, helping to generate coherent outputs to peripheral clocks throughout the body. In particular, arginine vasopressin (AVP)– and vasoactive intestinal peptide (VIP)–expressing neurons have been recognized as crucial subpopulations within the SCN and have thus been the focus of many chronobiological studies. Here, we analyze the neuropeptide expression of 2 popular transgenic mouse strains commonly used to direct or restrict Cre-mediated recombination to AVP- and VIP-ergic neurons. The Avp-IRES2-Cre (JAX #023530) and Vip-IRES-Cre (JAX #010908) “driver” mouse strains express the Cre recombinase under the control of the endogenous Avp or Vip gene, respectively, allowing scientists either to ablate their gene of interest or to overexpress a transgene in a cell type–specific manner. Although these are potentially very powerful tools for chronobiologists and other scientists studying AVP- and VIP-ergic neurons, we found that neuropeptide expression in these mice is significantly decreased when an IRES(2)-Cre cassette is inserted downstream of the neuropeptide-encoding gene locus. The impact of IRES(2)-Cre cassette insertion on neuropeptide expression may be a confounding factor in many experimental designs. Our findings suggest that extreme caution must be exercised when using these mouse models to avoid misinterpretation of empirical results.

Reduced VIP Expression Affects Circadian Clock Function in VIP-IRES-CRE Mice (JAX 010908)

“…Of note, the VIP-IRES-Cre model was created using methods designed to minimize interference with native protein expression (i.e., knock-in allele inserted after 3′ stop codon including IRES sequence). Nevertheless, we show here that VIP is decreased in the SCN of both homozygous and heterozygous VIP-Cre mice, the latter of which is in agreement with a recent study (Cheng et al, 2019). We build on this previous work by including homozygous VIP-IRES-Cre mice in our analyses, employing PER2::LUC assays of clock function, and testing the role of AVP signaling in this VIP-deficient model.…”

“…One advantage to examining this particular genetic model is that the phenotypes and consequences of VIP deficiency are well established. In agreement with a recent report (Cheng et al, 2019), we find that VIP expression is reduced by the VIP-IRES-Cre transgene in adult heterozygous VIP-Cre mice ( Vip cre/+ , JAX 010908). Further, we demonstrate that VIP is decreased at an earlier age and in a more pronounced manner in homozygous VIP-Cre mice ( Vip cre/cre ).…”

“…Technical advances have led to widespread use of this technique and refinements designed to mitigate off-targets effects (e.g., insertion of the IRES sequence). However, it has been noted that the transgene can interfere with gene expression even in the most well-constructed models (Cheng et al, 2019; Hoffmann et al, 2019; Taniguchi et al, 2011; Viollet et al, 2017).…”

Output from VIP cells of the mammalian central clock regulates daily physiological rhythms

“…We did, however, find that absolute amplitudes of VIP-ChR2 SCN firing rate rhythms were modestly reduced relative to control ( Supplementary Fig. 4e), perhaps reflecting the reduced VIP expression observed in VIP +/cre mice 41 . Notably, circadian rhythms in behaviour are not noticeably impaired in this line, a finding which is consistent with our own observations that the basic rhythmic properties are intact in VIP-ChR2 slices.…”

Suprachiasmatic VIP neurons are required for normal circadian rhythmicity and comprised of molecularly distinct subpopulations

“…While our physiological and behavioral recordings in the VIP cre/cre mouse are potentially consistent with a hypomorphic allele, the expression of VIP transcript (per RNAscope in situ hybridization) and number of VIP+ cell bodies (per GFP+ cells in the genetic cross) within the SCN did not appear to be appreciably altered in the homozygous state. It is nevertheless possible, however, that less VIP was being produced in the SCN of the homozygous VIP cre/cre mouse, as has been reported before in other targeted transgenic IRES-Cre mouse lines 21 , and as was clearly the case for the Vip-IRES-Cre (JAX #010908) mouse line that has been employed in a number of previous chronobiology studies 51 . If it were the case that less VIP was produced in the SCN of our mice, however, the mild phenotypes observed in the hypomorph would only emphasize the importance of VIP in proper SCN network function (i.e., other SCN cell populations are apparently unable to fully compensate for the presumptive decrease in VIP production).…”