Deletion polymorphism of Disc1 is common to all 129 mouse substrains: implications for gene-targeting studies of brain function

Abstract

We report that the Disc1 gene in all extant 129 mouse inbred substrains has a deletion, previously considered specific to the 129S6/SvEv substrain, which is predicted to abolish production of the full-length protein. This finding has implications for the study of knockout mice generated from 129-derived embryonic stem cells.

Figure 1.—

PCR genotyping assay to detect the Disc1 deletion. C57BL/6 mice have a wild-type Disc1 allele, while 129S6 mice have a 25-bp deletion (dots) in exon 6 of Disc1. The forward primer (5′-GCT GTG ACC TGA TGG CAC T-3′) within exon 6 corresponds to nucleotides 128,021,467–128,021,485, and the reverse primer (5′-CTT CTC ACC TGA GCA CAC CT-3′) within intron 6–7 is the reverse complement of nucleotides 128,021,642–128,021,662 of chromosome 8. Physical map distances between the primers were obtained using the UCSC Genome Browser (http://genome.ucsc.edu/; mouse assembly February 2006). The primers are designed to amplify a fragment of 171 bp from 129S6 and a fragment of 196 bp from C57BL/6. Each 25-μl PCR contains 17.7 μl nuclease-free water, 2.5 μl 10X PCR buffer [500 mm KCl, 100 mm Tris–HCl (pH 9.0 at 25°), 1% Triton X-100], 1.6 mm magnesium chloride, 0.2 mm each of dATP, dTTP, dCTP, and dGTP, 0.5 μm of each primer, 1.5 units Taq DNA polymerase, and 50 ng genomic DNA. Reactions are incubated at 94° for 5 min, followed by 35 cycles of 94° for 20 sec, 54° for 1 min, and 72° for 40 sec, followed by 72° for 10 min.

Figure 2.—

Disc1 deletion genotyping gel. M, 100-bp DNA ladder (1.0 μg; 70 ng per band). B6, C57BL/6J. S6, 129S6. F1, B6129F1. P1, 129P1. P2, 129P2. P3, 129P3. P4, 129P4. S1, 129S1. S2, 129S2. S4, 129S4. S5, 129S5. S7, 129S7. S8, 129S8. T1, 129T1. T2, 129T2. X1, 129X1. Following thermocycling, 20 μl of PCR product were electrophoresed at 8.5 V/cm through a 3% (w/v) agarose gel in 1X TAE buffer (40 mm tris-acetate, 2 mm EDTA) for 75 min. The sizes of the DNA ladder fragments and PCR products are indicated. The extra, upper band in the F1 (heterozygote) lanes is a heteroduplex of the wild-type and mutant strands, in which the 25-bp mismatch of the deletion forms a loop that slows migration through the gel.

Figure 3.—

Sequence chromatograms showing the 25-bp deletion of Disc1 in representatives of the steel (129S6), parental (129P2), Ter (129T1), and X (129X1) lineages of the 129 inbred strain family. An asterisk indicates the location of the deletion in the 129S6 sequence. Alignment and comparisons with the wild-type C57BL/6J sequence were performed using Sequencher software (Gene Codes, Ann Arbor, MI).