| First Author | Peters J | Year | 1995 |
| Journal | Mouse Genome | Volume | 93 |
| Issue | 4 | Pages | 1041-3 |
| Mgi Jnum | J:30570 | Mgi Id | MGI:78681 |
| Citation | Peters J, et al. (1995) Genetic and phenotypic analysis of Del(1)Spr3H and Pax3Sp-4H. Mouse Genome 93(4):1041-3 |
| abstractText | Full text of Mouse Genome contribution: GENETIC AND PHENOTYPIC ANALYSIS OF Del(1)Spr3H AND Pax3Sp-4H. Jo Peters, Peter Glenister, Heather Moseley and Ann Roberts. Mammalian Genetics Unit, Medical Research Council, Chilton, Didcot, Oxon OX11 ORD INTRODUCTION The deletion Del(l)Spr3H was recovered after X-irradiation and is characterised by growth retardation, ventral white spotting in most animals, and occasional head dots (1). The deletion extends for several centimorgans on Chromosome 1, (Chr 1), encompasses Pax3, and has a proximal breakpoint between Tnpl and Vil and a distal breakpoint between Akp3 and Acrg (2). The deletion is cytologically visible with breakpoints in 1C4 resulting in the loss of 2% of Chr 1 (3). Pax3Sp-4H was also recovered following X-irradiation and has a similar white spotting phenotype to Del(l)Spr3H (4). Pax3Sp-4H involves deletion of the entire Pax3 gene (5), but flanking genes are not deleted, and the deletion is not cytologically visible. We have further defined the extent of the deletions in Del(1)Spr3H and Pax3Sp-4H using SSR markers predicted to map to the region, and have examined the white spotting phenotypes associated with Del(1)Spr3H and Pax3Sp-4H on different backgrounds. Del(1)Spr3H will be referred to as Spr, and Pax3Sp4H as Sp4H for the rest of this paper. MATERIALS AND METHODS All stocks of mice were obtained from the colony in the Mammalian Genetics Unit, Chilton, UK. Both Sp4H and Spr were re-established from the Mammalian Genetics Unit Frozen Embryo Bank and were maintained by routinely crossing to (C3H/HeH x 101/H)F1 hybrids and selecting offspring with white belly spots. Fl hybrids with M. spretus were generated by in vitro fertilization, as natural matings were unsuccessful. DNA samples: Initially DNA was extracted from lung and spleen using standard procedures, but subsequently DNA was prepared from tail and spleen essentially as described in (6). PCR amplification: Seven loci, known to map in the region of Pax3, identified as simple sequence repeats (SSRs) and defined by a specific pair of primers, were analysed by the polymerase chain reaction. The loci are listed in Table 1. Primers were purchased from Research Genetics (Huntsville, AL., USA). Primer sequences, together with allelic size variations were obtained from (7). SSRs were amplified from 120 ng template DNA in a final reaction volume of 20ul containing 20mM Tris-HCl pH 8.0, 50mM Kcl, 320uM DTT, 0.04% Wl, 0.l mg/ml BSA, 165uM each primer, 0.2U/ml Taq polymerase (Boehringer), and 1.5mM MgCl2. PCR reactions were carried out in a Perkin-Elmer thermocycler. Following an initial denaturation at 95 degrees C for 5 min, the reaction mixtures underwent 30 cycles of 94 degrees C for 90 s, 55 degrees for 90 s, 72 degrees C for 90 s, followed by a final extension at 72 degrees C for 9 min. Amplified products were resolved on Visigel separation matrix (Stratagene) using the conditions described by the manufacturer, and visualized by ethidium bromide staining. RESULTS AND DISCUSSION Deletion of SSR markers The allele sizes of all the SSRs differed in C3H/HeH and 101/H and three, D1Mit19, D1Mit10, and D1Mit26 were heterozygous in at least one proven Spr/+ mouse, indicating that they lay outside the deletion (Table 1). A third allele for all the SSRs was present in M.spretus and D1Mit8 was heterozygous in a (Spr/+ x M.spretus)F1 hybrid, so that this marker must also lie outside the deletion. For three of the SSRs, D1Mit23, D1Mit24 and D1Mit46 all Spr/+ mice tested were apparently homozygous for either the 101/H or C3H/HeH allele. The Spr/+ mice were then crossed as appropriate to C3H/HeH or 101/H, and progeny were scored for the markers. Spr/+ mice carried either the C3H/HeH or 101/H alleles (depending on the cross) whereas +/+ sibs were heterozygous for both the C3H/HeH and 101/H alleles signifying that these three markers lay within the deletion. Thus, from the results with SSRs, the proximal breakpoint of the Spr deletion lies between D1Mit19 and D1Mit23 and D1Mit24, (the latter two markers have not been separated by recombination), and the distal breakpoint between D1Mit46 and D1Mit8. D1Mit19 must be proximal to Vil, and D1Mit8 distal to Akp3, the most proximal and distal gene markers respectively within the Spr deletion (2), (Fig 1). Neither D1Mit19 nor D1Mit8 had been been mapped previously in relation to known genes. The results accord well with composite maps of Chr 1 (8, 9). All seven SSRs were heterozygous in (Sp4H/+ x M.spretus)Fl hybrids denoting that none are deleted in Sp4H (Table 1, Fig 1). These data add to earlier findings where only Pax3 was found to be deleted (5), suggesting that Sp4H is a small deletion. Table I. (Legend). Deletion analysis of Spr and Sp4H. Allele sizes of SSRs found in stocks from the Mammalian Genetics Unit, measured after separation on Visigel are shown. Abbreviations for mouse strains are 101 for 101/H, C3H for C3H/HeH and MSP for Mus spretus. > denotes an allele is longer than another. The distance of loci from the centromere has been taken from reference 8. Locus: D1Mit19; Distance from centromere (cM): 36.9; Size of largest allele (bp): 390; Size variation: MSP>C3H>101; Deleted in Spr: No; Deleted in Sp4H: No. Locus: D1Mit23; Distance from centromere (cM): 41; Size of largest allele (bp): 220; Size variation: MSP>101>C3H; Deleted in Spr: Yes; Deleted in Sp4H: No. Locus: D1Mit24; Distance from centromere (cM): 41; Size of largest allele (bp): 210; Size variation: 101>C3H>MSP; Deleted in Spr: Yes; Deleted in Sp4H: No. Locus: D1Mit46; Distance from centromere (cM): 43.1; Size of largest allele (bp): 410; Size variation: MSP>C3H>101; Deleted in Spr: Yes; Deleted in Sp4H: No. Locus: D1Mit8; Distance from centromere (cM): 52; Size of largest allele (bp): 218; Size variation: 101>C3H>MSP; Deleted in Spr: No; Deleted in Sp4H: No. Locus: D1Mit10; Distance from centromere (cM): 56.6; Size of largest allele (bp): 160; Size variation: MSP>C3H>101; Deleted in Spr: No; Deleted in Sp4H: No. Locus: D1Mit26; Distance from centromere (cM): 59.7; Size of largest allele (bp): 230; Size variation: MSP>C3H>101; Deleted in Spr: No; Deleted in Sp4H: No. Fig. 1. (Legend). Genetic map showing extent of deletions. Morphological phenotype Sp is characterised by white spotting on the belly, feet, tail and occasionally the head. Neither (Spr/+ x M.spretus)F1 nor (Sp4H/+ x M.spretus)F1 hybrids showed white belly spotting, and low grade spotting on the feet and tail occurred rarely; consequently these mice were scored for Sp by crossing to C3H/HeH and looking for progeny with ventral white spots. Both Spr/+ and Sp4H/+ showed different levels of expression in crosses to C3H/HeH and 101/H. Sp/+ offspring arising from the crosses (Spr/+ x 101/H) and (Sp4H/+ x 101/H) rarely showed the complete phenotype and frequently showed only low grade spotting on the feet and tail. By contrast, the full phenotyoe was often seen in Sp/+ offspring arising from the crosses (Spr/+ x C3H/HeH) and (Sp4H/+ x C3H/HeH) (Table 2). Thus, overall, the penetrance of Spr and Sp4H appeared greatest on a C3H/HeH and least in M.spretus. Table 2. Spotted mice arising in crosses with Spr and Sp4H. Cross: 1; Cross: Spr/+ x 101; No. of mutant mice with full phenotype: 6 (12%); No. of mutant mice with low grade spotting only: 46 (88%); No. of wild type mice: 64; Ratio of spotted: +: 0.8:1; % of total with full phenotype: 5; % of total with low grade spotting only: 40. Cross: 2; Cross: Sp4H/+ x 101; No. of mutant mice with full phenotype: 2 (4%); No. of mutant mice with low grade spotting only: 48 (96%); No. of wild type mice: 75; Ratio of spotted: +: 0.7:1; % of total with full phenotype: 1.6; % of total with low grade spotting only: 38. Cross: 3; Cross: Spr/+ x C3H; No. of mutant mice with full phenotype: 34 (52%); No. of mutant mice with low grade spotting only: 32 (48%); No. of wild type mice: 59; Ratio of spotted: +: 1.1:1; % of total with full phenotype: 27; % of total with low grade spotting only: 26. Cross: 4; Cross: Sp4H/+ x C3H; No. of mutant mice with full phenotype: 32 (94%); No. of mutant mice with low grade spotting only: 2 (6%); No. of wild type mice: 63; Ratio of spotted: +: 0.5:1; % of total with full phenotype: 33; % of total with low grade spotting only: 2. Whereas in crosses with Spr the ratio of spotted to wild type progeny is close to the expected 1:1 there appears to be a shortage of spotted mice arising from crosses involving Sp4H. Post-natal losses were unexceptional (7%). A similar shortage had been reported in a previous study (4), in which neither significant pre- nor post-natal losses of Sp4H/+ occurred. Possibly Sp4H is less penetrant than Spr, but further investigations have not been undertaken and the reasons for the shortage are not clear. Table 3. D1Mit46 is a marker for Spr. Origin of D1Mit46 allele: C3H; No. of mice with ventral spotting: 12; No. of mice with low grade spotting: 11; Wild type: 0. Origin of D1Mit46 allele: C3H and 101; No. of mice with ventral spotting: 0; No. of mice with low grade spotting: 2; Wild type: 24. 49 of the progeny (both Sp/+ and +/+) arising from the mating of (Spr/+101/H x +C3H/+C3H), (Table 2, cross 3), were typed for D1Mit46, which lay within the Spr deletion and showed a clearly resolvable difference in allele size between 101/H and C3H/HeH. 12 mice with the full phenotype carried only the C3H/HeH derived allele of D1Mit46 indicating they were indeed Spr/+C3H. 24 apparently wild type mice were heterozygous for the C3H/HeH and 101/H derived alleles of D1Mit46 showing that they did not carry Spr. 11/13 mice with low grade white spotting only were Spr/+C3H, for they were hemizygous for the C3H/HeH derived allele of DlMit46. Two with markings limited to toes did not carry Spr for they were heterozygous for D1Mit46 (Table 3). Thus, the complete phenotype was completely reliable as an indicator of Sp, but low grade white spotting was not. ACKNOWLEDGMENTS: This work was supported by a project grant from the MRC HGMP directed programme. REFERENCES 1. Beechey, C.V. and Searle, A.G. (1986). Mouse News Lett. 75:28. 2. Epstein, DJ. et al. (1991a). Genomics 10:89-93. 3. Cattanach, B.M. et al. (1993a). Nature Genetics 3:56-61. 4. Cattanach, B.M. et al. (1993b). Mouse Genome 91:115-116. 5. Goulding, M. et al. (1993). Genomics 17:355-363. 6. Whitelaw, C.B.A. et al. (199l). Transgenic Research 1:3-13. 7. Dietrich, W.F. et al. (1994). Nature Genetics 7 special issue:220-245. 8. Seldin, M.F. (1994). Mammalian Genome 5:Sl-S21. 9. Lyon, M.P. and Kirby, M.C. (1994). Mouse Genome 92:19-61. |
