Mice from the standard inbred strains C57BL/6 J (JAX stock #000664) and BALB/cJ (JAX stock #000651), and co-isogenic BALB/cJ-squig/GrsrJ mice (JAX stock #026620) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The squig mutation was maintained at CCSU by crossing heterozygotes with mutant homozygotes. Mutants were reliably identified (with at least 99.3% penetrance in our colony) by their short and curly tails that are apparent from birth (see Additional file 1: Figure S1). At the end of the study, mice were killed by cervical dislocation or by use of CO2 gas added to a chamber (typically their home cage) using a compressed gas cylinder fitted with a flow meter adjusted to displace only 30–70% of the chamber volume per minute (consistent with the recommendations of the AVMA Guidelines for the Euthanasia of Animals, 2020 Edition). Only the P.I. (TRK) who was trained at The Jackson Laboratory (Bar Harbor, ME) and has over 30 years of experience, performed euthanasia.
DNA isolation and marker typing
Genomic DNA was isolated from 2 mm tail-tip biopsies taken from two- to three-week-old mice using NucleoSpin® Tissue kits (Macherey–Nagel, Düren, Germany; distributed by Clontech Laboratories, Inc., Mountain View, CA, USA), as directed. DNA samples from standard inbred and mutant strains that we do not routinely maintain in our colony were purchased from The Jackson Laboratory’s Mouse DNA Resource.
The polymerase chain reaction (PCR) was performed in 13 µl reactions using the Titanium® PCR kit from Clontech Laboratories, as directed. Oligonucleotide primers for PCR were designed and synthesized by Integrated DNA Technologies, Inc. (Coralville, IA, USA), based on sequence information available online [2, 3]. To score PCR product sizes for dimorphic microsatellite markers, reactions plus 3 µl loading buffer were electrophoresed through 3.5% NuSieve® agarose (Lonza, Rockland, ME, USA) gels. Gels were stained with ethidium bromide and photographed under ultraviolet light. In addition to eight standard microsatellite markers  on Chr 11, eight DNA markers based on single nucleotide polymorphisms previously reported to differ between strains BALB/cJ and C57BL/6 J [2, 3] were scored. These markers (herein designated SNP#) are described in detail in Additional file 2: Table S1 and Additional file 3: Table S2.
For DNA sequence analysis, about 1.5 µg of individual PCR amplimers were purified and concentrated into a 30 µl volume using NucleoSpin® PCR Clean-up kits, and then shipped to the Keck Foundation Resource Laboratory at Yale University (New Haven, CT, USA) for primer-extension analysis.
A “3-primer” test for detecting Meox1squig
To rapidly determine genotypes at the squig locus (especially among phenotypically wild type mice) we used a standard PCR assay that employed three primers: a single forward primer (F1, 5’-GTTACCAGGAGGTGCTCAAA-3’) that annealed 5’ to the Meox1 deletion and two reverse primers—one that annealed within the Meox1 deletion (R1, 5’-GTGAAATGTGAGAGAGGAGAGG-3') and one that annealed 3’ to the deletion, within Gm11551, Exon 2 (R2, 5’-CCAGATCCCAGCAATCAAGATA-3'). Primers F1 and R1 direct the amplification of a 268 bp product specific to wild type BALB/cJ templates; the F1, R2 primer pair direct the amplification of a 456 bp product specific to squig templates.