Genotype, phenotype, and karyotype correlation in the XO mouse model of Turner Syndrome

Abstract

The murine model for Turner Syndrome is the XO mouse. Unlike their human counterparts, XO mice are typically fertile, and their lack of a second sex chromosome can be transmitted from one generation to the next as an X-linked dominant trait with male lethality. The introduction of an X-linked coat-color marker (tabby) has greatly facilitated the maintenance of this useful mouse strain. XO mice can be produced in large numbers, generation after generation, and rapidly identified on the basis of their sex and coat color. Although this breeding scheme appears to be effective at the phenotype level, its utility has never been conclusively proved at the molecular or cytogenetic levels. Here, we clone and sequence the tabby deletion break point and present a multiplex polymerase chain reaction-based assay for the tabby mutation. By combining the results of this assay with whole-chromosome painting data, we demonstrate that genotype, phenotype, and karyotype all show perfect correlation in the publicly available XO breeding stock. This work lays the foundation for the use of this strain to study Turner Syndrome in particular and the X chromosome in general.

Figure 1

Phenotypes of animals in the XO breeding stock. To maintain the XO breeding stock, an agouti male from an outside colony (I-1; Eda⁺/Y, XY) is bred to a presumed XO female (with tan-colored fur and bald spots behind the ears) from the XO breeding stock (I-2; presumed Eda^Ta/O, XO). Three types of offspring are seen: female animals with stripes of agouti fur and stripes of tan-colored fur (II-1; presumed Eda⁺/Eda^Ta, XX), male animals with tan-colored fur and bald spots behind the ears (II-2; presumed Eda^Ta/Y, XY), and female animals with agouti fur (II-3; presumed Eda⁺/O, XO). Animals II-2 and II-3 are interbred, and there are again 3 different kinds of progeny: female animals with stripes of agouti fur and stripes of tan-colored fur (III-1; presumed Eda⁺/Eda^Ta, XX), male animals with agouti fur (III-2; presumed Eda⁺/Y, XY), and female animals with tan-colored fur and bald spots behind the ears (III-3; presumed Eda^Ta/O, XO). Animal III-3 can then be bred to another agouti male from an outside colony to propagate the stock indefinitely. The sex of each animal is shown at the bottom right of each photograph (♂ = male, ♀ = female). All phenotypings (both sex and coat color) are done by simple visual inspection. The photographs also demonstrate that the hemizygous Eda^Ta mutants (animals I-2, II-2, and III-3) have a tendency to squint, which is likely secondary to well-documented deficiencies in the glands surrounding the eyes in mutant animals (Grüneberg 1971). Note that Eda^Ta/O, XO females (I-2 and III-3) are bred to Eda⁺/Y, XY males from an outside colony (I-1; a male from JAX stock number 001201) as opposed to Eda⁺/Y, XY males from within the XO breeding colony (III-2). This is done to promote fertility within the colony as the stock is difficult to maintain if brother–sister matings are used exclusively (Deckers and van der Kroon 1981).

Figure 2

Eda genotyping. (a) The Eda^Ta mutation was cloned and sequenced and found to be a pure 1073-bp deletion that removes the entire first exon of the Eda gene (GenBank accession number EU178100). In a multiplex PCR reaction, primers A and B amplify a 216-bp product from wild-type DNA, whereas primers C and D amplify a 162-bp product. The Eda^Ta deletion removes the annealing sites for primers B and C and brings together the annealing sites for primers A and D, yielding a single 168-bp product. Thus, DNA from a homozygous or hemizygous wild-type animal yields 216-bp and 162-bp PCR products, DNA from a homozygous or hemizygous mutant animal yields a single band of 168 bp, and DNA from a heterozygote yields all 3 bands. The 3 different PCR products were all sequenced to confirm their identities (data not shown). (b) The pedigree shows a graphic representation of the breeding scheme shown in Figure 1. Males are shown as squares, and females are shown as circles. Dark brown shading indicates an agouti coat color, tan shading indicates a tan-colored coat color (and bald spots behind the ears), and stripes indicate a striped coat. The electrophoresis gel shows the results of the Eda multiplex PCR reaction for each of the respective animals in the pedigree from Figure 1. All animals have the expected genotypes, and there is perfect genotype–phenotype correlation. DNA from agouti animals yields 216-bp and 162-bp bands (Eda⁺), DNA from tan-colored animals (with bald spots behind the ears) yields a single band at 168 bp (Eda^Ta), and DNA from striped animals yields all 3 bands (Eda⁺/Eda^Ta). The assay is not quantitative and does not directly distinguish homozygotes (Eda⁺/Eda⁺ and Eda^Ta/Eda^Ta) from hemizygotes (Eda⁺/Y, Eda⁺/O, Eda^Ta/Y, and Eda^Ta/O). However, it demonstrates that animal I-2 fails to transmit her Eda^Ta allele to her female agouti offspring (II-3), and animal II-3 in turn fails to transmit her Eda⁺ allele to her offspring with tan-colored fur and bald spots behind the ears (III-3), which is consistent with all 3 animals having the XO karyotype.

Figure 3

Whole-chromosome painting. Whole-chromosome painting of the X and Y chromosomes was performed on chromosome spreads from bone marrow cells of the animals from generations II and III in Figures 1 and 2. The DAPI counterstain gives all chromosomes a blue fluorescent background color. The cyanine 3–labeled X chromosome probe produces a red fluorescent signal, and the fluorescein isothiocyanate-labeled Y chromosome probe produces a green fluorescent signal. Cells from animals II-1 and III-1 have 2 chromosomes with red signals (XX), and cells from animals II-2 and III-2 have 1 chromosome with a red signal and 1 with a green signal (XY), whereas cells from animals II-3 and III-3 have 1 chromosome with a red signal and no second sex chromosome signal (XO).