Twenty-seven Single-copy Site-specific Cre-Driver Mouse Strains for Advancing Eye and Brain Research

Abstract

Purpose : Cre-driver mice (mice that express cre in a temporal and region or cell-type specific way) are important tools for basic and preclinical research. Large-scale efforts such as the International Knockout Mouse Consortium, which aims to develop mutations in all protein-coding genes, depends upon inactivation of their conditional alleles by cre-recombinase. However, the cre-driver mouse resource is relatively small, and random-insertion cre-driver mice may show unpredictable expression due to copy number and genomic position effects. In this work, we have used single-copy site-specific knock-ins to generate a large number of mouse strains carrying either a BAC-based “MaxiPromoter” (MaxiP) driving a tamoxifen-inducible improved cre (icre), or plasmid-based “MiniPromoter” (MiniP) driving an inducible first, constitutive icre allele. This new resource is focused on cre expression in the eye and the brain.

Methods : Both the MaxiPs and MiniPs driving icre/ERT2 or icre/frt/ERT2/frt respectively were cloned into Hprt homologous-recombination targeting vectors. Using C57BL/6N embryonic stem cells (mEMS4855/4857), chimeras were generated, and germline offspring tested for expression by RT-PCR in key tissues. A subset of 2 MaxiP and 4 MiniP strains were bred to a cre-reporter tdTomato strain (JAX 007914). Adult offspring were fed a diet of tamoxifen food for 4 weeks, and were processed 3 weeks later to allow for expression. In addition, the MiniP strains were crossed with a Flpo-driving strain (MMRRC 032247) creating a constitutive icre allele, and adult mice were processed. Animals were perfused and tissues cryosections and examined for tdTomato epifluorescence.

Results : In total, 10 MaxiP-icre-ERT2 strains and 17 MiniP-icre/ERT2 strains were established. RT-PCR showed positive expression of icre-ERT2 in relevant tissues for all 27 strains. Histological examination of 10 strains, including both inducible and constitutive icre alleles, showed expression in, for example, retinal bipolar and Müller glia cells, as well as the cornea.

Conclusions : This resource of 27 mouse strains demonstrates the feasibility of single-copy site-specific knock ins for the generation of substantial numbers of cre-driver strains. In addition, it provides a set of important tools for basic and preclinical eye and brain research. Finally, all these strains are available to the community through The Jackson Laboratory.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.