Luxate-like 2 (Lxl2), a remutation to Luxate (Lx).
Patricia F. Ward-Bailey, Belinda S. Harris, Leah Rae Donahue, Rod T. Bronson, Kenneth R. Johnson, and Muriel Davisson
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 Symbol: Lxl2
Mutation Name: Luxate-like 2
Strain of origin: AKR/J
Current strain name: B6;AKR-Lxl2/J
Stock# 004502 (view JAX® Mice Data Sheet for additional information including Price and Supply Information)
Phenotype category: limbs
Abstract
Luxate like 2 (Lxl2) is a remutation to luxate (Lx). Lxl2 arose in a production colony of AKR/J mice at The Jackson Laboratory in 1998. This dominant mutation causes the animal to present with all four limbs at odd angles to the body. There are extra toes on all four limbs and the rear legs are oriented backward. Severe arthritis of the knee was observed in one homozygous female, but no other histological lesions were seen. We used a backcross to map the Lxl2 mutation to Chr 5. The most likely gene order places the mutation between D5Mit1 and D5mit13 in 53 tested meioses. Luxate-like 2 could not be directly tested for allelism by complementation analysis with luxate because both are dominant; however, its identical phenotype and genetic map position indicate allelism.
Origin and Description
The Lxl2 remutation was found at The Jackson laboratory in 1998 by Jennifer Haycoch in a pooled mating in a colony of AKR/J mice (JR # 648) in a production colony in Room MP14.
The phenotype when heterozygote shows extra toes on all four feet and the rear legs twisted backward. It is believed that homozygous Lxl2 mutants die in utero because a homozygous mutant has never been born. When hetrozygotes are mated together, and the resulting Lxl2/? animals are progeny tested, only Lxl2/+ are found and no homozygotes are produced.
This remutation is being transferred to a C57BL/6J background for maintenance because the breeding was poor on the AKR/J background. Currently it is at N2 on C57BL/6J. DNA was saved from the original mutant background.
Mutants are easily identified at birth by their twisted rear legs and extra digits on all four limbs. The Lxl2 mutation is maintained by mating a female heterozygote to a +/+ male. Males heterozygous for the Lxl2 mutation do not always breed.
Phenotypic Characterization
X-ray image of whole mouse clearly shows twisted limbs and extra digits characteristic of Lxl2/+ mutants.
Histological techniques. Tissues for histopathological examination were prepared from both Lxl2/+ and control animals. Tissues were removed from animals deeply anesthetized with tribromoethanol (Avertin) and fixed by intracardiac perfusion of Bouin’s solution following a flush of saline. After demineralization in Bouin’s, cross sections of spine with spinal cord in situ, and midsagittal sections of hind leg were prepared. Cross sections of brain and most somatic organs were prepared. Organs sampled were eyes, pituitary, salivary glands, thyroids, lung, heart, thymus, liver, spleen, pancreas, stomach, small intestine, colon, cecum, kidneys, adrenals and reproductive organs. Sections of all tissues were stained with hematoxylin and eosin (H&E). Brain and spinal cord sections were also stained with luxol-fast blue-cresylecht violet (LFB-CV) No lesions were observed in any organ. Specimens from 2 Lxl2 and 2 +/? mice were cleared and stained with alizarin red S and alcian blue to demonstrate bone and cartilage (Green, M.C., 1952). Apart from the twisted rear legs and extra digits on all four limbs, no abnormalities were observed.
ABR threshold tests (Zheng 1999) of four 7-week-old and two 40-week-old mutants indicated normal hearing.
Genetic Analyses
Lxl2 inheritance as a dominant mutation was shown by outcrosses to both C57BL/6J and CAST/Ei. In both outcrosses, affected animals were seen in the F1 generation.
For linkage analysis, a backcross was utilized to produce mutant mice. The offspring of a cross between a female AKR/J mutant and a male C57BL/6J were crossed back to C57BL/6J +/+ animals. The backcross progeny were scored visually for phenotype and spleens and tails tips from 53 mutant animals were collected and stored at -70C for subsequent DNA typing to map the mutation. DNA was extracted from the frozen tail tips of 53 mutant produced in the linkage cross by a standard hot sodium and Tris (HotSHOT) procedure (Truett,et al., 2000). PCR primer pairs (MapPairs, Research Genetics, Huntsville Ala.) of microsatellite markers D5Mit47, D5Mit70, D5Mit1, D5Mit349, D5Mit386, D5Mit387, D5Mit13, D5Mit255, and D5Mit205 were used to localize the mutation on Chr 5. 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).
Markers on Chr 5 were chosen first for linkage analysis because this mutation causesa phenotype that looks like Luxate (Lx). Lxl2 mapped to the same position on Chr 5 as Lx. The gene order and recombination estimates with standard errors, calculated with the Map Manger computer program (Manley 1993), are centromere-[D5Mit47, D5Mit70]-3.85+/- 2.67-[D5Mit1]-7.69+/-3.70-[D5Mit349, D5Mit386, D5Mit387, Lxl2]-3.03+/- 2.98-[D5Mit13]-6.45+/- 4.41-[D5Mit255].
Acknowledgments
We thank the following for their excellent technical expertise: Coleen Marden, Jane Maynard, Heping Yu, Qing Yin Zheng, Norm Hawes
References Cited
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