Chocolate-like, a spontaneous mutation causing a dark brown coat color in the mouse.
Patricia F. Ward-Bailey MS, Belinda Harris BS, Rod Bronson DVM, Ken Johnson Ph.D, and Muriel T. Davisson Ph.D
Source of Support: This research was supported by NIH/NCRR grant RR01183 to the Mouse Mutant Resource (M.T. Davisson, PI) and Cancer Center Core Grant CA34196.
Mutation (allele) name: chocolate-like
Mutation (allele) symbol: chtl
Gene symbol: chtl
Strain of origin:B6.129P2-Nos2tm1Lau
Current strain name:B6(129P2)-Nos2tm1Lau-chtl/J
Stock #:004684 (view JAX® Mice Data Sheet for additional information including Price and Supply Information)
Phenotype categories: coat color
Origin and Description
The chocolate-like mutation was found at The Jackson Laboratory in 1998 in the B6.129P2-Nos2tm1Lau strain at generation N7F4 + F16. This mutant has an overall lighter coat, tail, and ears than a black a/a control. Both females and males breed and it is maintained as a homozygous stock. Stock # 002609-B6.129P2-Nos2tm1Lau is the background control. This recessive mutation, which we have named chocolate-like (gene symbol, chtl), maps to Chr 7 between the markers D7Mit211 and D7Mit321 and is non recombinant with D7Mit31. Based on linkage map position and Ensembl location for the markers used, it was thought that this was a remutation to chocolate (cht). A test for allelism with cht however did not produce the expected chocolate colored mice, but instead produced a dull grey phenotype.
Pathology
A routine pathological screen of one female and one male chtl/chtl mice at 11 weeks of age showed no lesions.
Hearing tests. Hearing was assessed by ABR threshold analysis (Zheng et al. 1999) of 2 chtl/chtl mice tested at 4 months of age. The ABR results showed that the homozygous mutants had normal hearing.
Eye Examination. The eyes of two homozygous mutants were examined with an opthalmascope and were determined to be normal.
Genetic anaylsis
chtl is inherited as a recessive mutation as shown by segregation in the traditional linkage cross analysis described below. The progeny produced showed no visible mutants in the F1 generation (0/26) and segregation in the F2 (23 chtl/398 total progeny) produced 5.78% mutants, less than the expected 25%.
For linkage analysis, an intercross was utilized to produce mutant mice. A CAST/Ei female was mated to a B6.129P2-Nos2tm1Lau homozygous mutant male. F1 hybrids from this initial cross were then intercrossed to produce the F2 progeny. The F2 progeny were scored visually for phenotype and spleens and tail tips from 21 homozygous mutant animals were collected and stored at -70C for subsequent DNA typing to map the mutation.
DNA isolation. DNA was extracted from the frozen tail tips of 21 mutant (homozygous) F2 mice produced in the linkage cross by a standard Hot Sodium and Tris (HotSHOT) procedure (Truett,et al., 2000).
Polymerase chain reaction. PCR primer pairs (MapPairs, Research Genetics, Huntsville Ala.) of microsatellite markers D7Mit230, D7Mit211, D7Mit313, D7Mit321, D7Mit43, and D7Mit259 were used to localize the mutation on Chr 7. PCR analyses were carried out in 10 ul total volume reactions containing 20 ng genomic DNA, by previously described methods (Ward-Bailey et al. 1996).
Mutation segregation ruled out Chromosome X linkage. A genome sweep of F2 progeny from the CAST intercross was begun by typing DNA samples from 21 homozygous mutant animals for segregating MIT microsatellite markers, starting with Chromosomes 7and 14, chosen because mutations with similar phenotypes ( cht and slaty) are located there. Linkage of chtl was first detected on Chr 7 with D7Mit43. DNA samples were then typed for five additional Chr 7 markers. The recombination estimates and best gene order are centromere-[D7Mit230]-5.06 +/-3.45-[D7Mit211]-7.65+/-4.16-[D7Mit31, chtl ]-2.40+/-2.35-[D7Mit321]-9.76+/-4.53-[D7Mit43]-17.43+/-5.75-[D7Mit259]. (See Map Figure)
Linkage analysis. Gene order and recombination frequencies were calculated with the Map Manager computer program (Manley), a MacIntosh program for storage and analysis of experimental mapping data. The complete Chr 7 linkage data for 21 F2 chtl/chtl mice have been deposited in the Mouse Genome Database, accession number J:82799.
Allelism tests
Chtl mice mated to DBA/2J mice was negative (all pups produced were a/a).
A test for allelism with homozygous cht mouse mated to a homozygous mutant mouse produced 32/32 progeny with a dull grey phenotype, unlike cht or a/a. These results suggested chtl is a new allele of the cht gene that produces a different phenotype from the original cht allele. When two matings of the grey color F1s from the allele test progeny were mated together, however, they produced segregating litters for a total of 10 cht looking mice and 3 normal black a/a mice. The appearance of the black mice means either the two mutations are pseudoalleles or modiefier genes can affect the phenotype expresssion.
Acknowledgements
We thank the following for their excellent technical expertise: Julie Seavey, Coleen Marden, Jane Maynard, Heping Yu, and Qing Yin Zheng.
References
Mouse Genome Database (MGD) Mouse Genome Informatics Project, The Jackson Laboratory, Bar, Harbor, Maine. World Wide Web (URL: http://www.informatics.jax.org).
Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, and Warman ML(2000) Preparation of PCR-Quality Mouse Genomic DNA with Hot Sodium Hydroxide and Tris (HotSHOT). Biotechniques 29:52-54.
Manley KF (1993) A MacIntosh program for storage and analysis of experimental mapping data. Mamm Genome 4,303-313.
Ward-Bailey PF, Johnson KR, Handel MA, Harris BH, Davisson MT. (1996) A new mouse mutation causing male sterility and histoincompatibility. Mamm. Genome 7, 793-797.
Zheng QY, Johnson KR, Erway LC(1999) Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses. Hear Res 130, 94-107.
