Mouse genetics

The novel alleles introduced in this work, Lrig1^T2A-iCreERT2, Lrig1^T2A-tdTomato, and Cdk6^T2A-td-sfGFP, were generated by conventional gene targeting procedure. Targeting vectors were constructed with homology arms subcloned from the RPCI-24 library of C57BL/6J mouse genomic DNA (CHORI). The vectors were electroporated into the G4 embryonic stem cells derived from 129S6/SvEvTac × C57BL/6Ncr F1 embryos [26]. The resulting germline mice were bred to Actb-FLPe or Pgk1-FLPo transgenic mice [27, 28] obtained from The Jackson Laboratory. Because the C57BL/6N mouse genome harbors a retinal degeneration mutation, rd8 [29], that may affect behavioral experiments, the mice were backcrossed to the C57BL/6J background while fixing the X and Y chromosomes to that background (the C57BL/6J mouse stock from The Jackson Laboratory). At the time of manuscript preparation, the three lines had been backcrossed for more than 8 generations to the C57BL/6J background.

The ROSA26^Ai14 mouse line [30] obtained from the Allen Institute for Brain Science was backcrossed to the C57BL/6J background for 7 generations by Simon Titen, Capecchi laboratory.

The Coffey Lrig1^creERT2 mouse line [19] obtained from The Jackson Laboratory was backcrossed to the C57BL/6J background for 4 generations. The Coffey Lrig1 allele is a null allele because creER^T2 cDNA completely replaces the first exon coding sequence which encodes Lrig1’s signal sequence to the plasma membrane.

The Lrig1^{T2A-iCreERT2/+}; ROSA26^Ai14/+ mice were bred to be genetically as similar as practically possible to each other: Lrig1^{T2A-iCreERT2/+} or Lrig1^{T2A-iCreERT2/T2A-iCreERT2} brothers were bred to ROSA26^Ai14/Ai14 sisters. Thus, the mice in experimental cohorts were not systematically randomized. Both sexes were included in the cohorts.

The targeting vectors and the mouse strains will be available from Addgene and The Jackson Laboratory.