Simultaneous deletion of floxed genes mediated by CaMKIIα-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Goα.

Chan-Il Choi,Lutz Birnbaumer,Sung-Soo Kim,Sang-Phil Yoon,Haeyoung Suh-Kim,Jung-Mi Choi,Young-Don Lee

doi:10.1038/emm.2014.14

Abstract

The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIα-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26+/stop-lacZ::CaMKIIα-Cre+/Cre mice generated by crossing CaMKIIα-Cre+/Cre mice with floxed ROSA26 lacZ reporter (Rosa26+/stop-lacZ) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26+/stop-lacZ::CaMKIIα-Cre+/Cre mice. Similarly, when double transgenic Gnao+/f::CaMKIIα-Cre+/Cre mice carrying a floxed Go-alpha gene (Gnaof/f) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (GnaoΔ) without inheriting the Cre transgene. The GnaoΔ/Δ mice closely resembled conventional Go-alpha knockout mice (Gnao−/−) with respect to impairment of their behavior. Thus, we conclude that CaMKIIα-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIα-Cre mice as breeding pairs.

Highlights

Heterotrimeric G proteins transduce numerous extracellular signals from receptors to intracellular signaling pathways
We show that when calmodulin-dependent protein kinase II alpha subunit (CaMKIIa)-Cre þ /Cre mice were crossed with floxed ROSA26 lacZ reporter (Rosa[26] þ /stop-lacZ) mice, the expression of lacZ was simultaneously induced by CaMKIIa-Cre in the brain and testis
Cre recombinase activity in CaMKIIa-Cre transgenic mice The CaMKIIa promoter was proven to drive a robust expression of Cre recombinase in the brain, its subregional expression varies in different CaMKIIa-Cre founder lines.[10,11,16,19]

Summary

Introduction

Heterotrimeric G proteins transduce numerous extracellular signals from receptors to intracellular signaling pathways. The Gi and Go proteins are activated by the same neurotransmitter receptors including D2 and D4, type 1 serotonin, M2 and M4 ACh, GABA-B and group 2 metabotropic glutamate receptors.[1] despite 70–85% identity, targeted inactivation of each of the Gia and Goa genes has markedly different consequences. Gia2-knockout mice show severe immunological deficits, including inflammatory bowel disease, and other immune abnormalities that precede an ulcerative colitis syndrome.[2] In contrast, deletion of Goa, which is abundantly expressed in the central nervous system, causes severe neurological deficits, such as hyperlocomotion, occasional seizure, hyperalgesia and loss of light response.[2] These distinctive behaviors point to a unique role of Goa in the brain

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Conclusion