A floxed transcription-disrupter (TD) ablates expression of the mouse Hcrtr2 gene in this OX2R TD conditional mutant strain. Cre-mediated excision of the TD restores expression under the direction of the native promoter.
Thomas Scammell, Beth Israel Deaconess Medical Center
The Hcrtr2 (hypocretin (orexin) receptor 2; also called OX2R) gene plays a role in regulating arousal, REM sleep, and depression-like behavior.
In this conditional mutant OX2R TD strain, a loxP-flanked transcription-disrupter (TD) cassette was introduced to intron 1 of the gene to prevent expression of functional protein. Cre-mediated excision of the TD restores expression under the direction of the native promoter.
The impact of this mutation was first tested electrophysiologically using in vitro patch-clamp recordings of histaminergic neurons of the tuberomammillary nucleus (TMN), a type of neuron that expresses only OX2R but not OX1R. Orexin-A ligand depolarizes TMN neurons from wild type mice but has no effect on TMN neurons from homozygous OX2R TD mice.
Behaviorally, OX2R TD mice had normal amounts of wake, non-REM, and REM sleep, but their wake bouts are short during the dark period. Cataplexy (episodes of sudden muscle atonia during active wakefulness) is rare in these mice. OX2R TD mice have milder sleep/wake deficits than prepro-orexin null mice. This phenotype is quite similar to that seen in constitutive OX2R null mice. These electrophysiological and behavioral deficits are fully restored after crossing OX2R mice with Zp3-Cre mice, which express Cre recombinase in the female germline.
OX1/2R TD progeny from crosses with OX1R TD mice (see Stock No. 027706) have severely fragmented sleep/wake behavior, similar to that seen in prepro-orexin null mice.
OX2R TD mice are useful as OX2R null mice in their native state. More importantly, they can be used to study the effects of focal or global restoration of OX2R signaling, using methods that produce focal or germline expression of Cre recombinase, respectively. Focal OX2R restoration can be used to test whether OX2R signaling in a specific brain region is sufficient to rescue specific behaviors.
A floxed transcription disrupter cassette was inserted into intron 1 via homologous recombination in 129-derived embryonic stem (ES) cells. The TD cassette consists of a mouse En2 splice acceptor site followed by an SV40 enhancer, neomycin resistance gene, and two HSV-TK poly(A) signals (a synthetic poly(A) signal/transcriptional pause signal, and another synthetic poly(A) signal followed by a Myc-associated zinc finger protein-binding site). Resultant founder mice were backcrossed to C57BL/6J mice for more than 15 generations by the donating laboratory.
|Allele Name||targeted mutation 1, Thomas E Scammell|
|Allele Type||Targeted (Conditional ready (e.g. floxed), No functional change)|
|Allele Synonym(s)||OX2R TD|
|Gene Symbol and Name||Hcrtr2, hypocretin (orexin) receptor 2|
|Strain of Origin||129|
|Molecular Note||A floxed transcription disrupter cassette was inserted into intron 1, 137 bp upstream of exon 2 via homologous recombination. The transcription disrupter cassette includes an engrailed 2 splice acceptor, SV40 enhancer, Neo, and a Myc-associated zinc finger protein-binding site. RT-PCR analysis confirmed the absence of full length transcript and the presence of a mutant mRNA.|
Homozygotes and heterozygotes are viable and fertile.
When using the Hcrtr2 null mouse strain in a publication, please cite the originating article(s) and include JAX stock #027707 in your Materials and Methods section.