Pleiotropic defects in ataxia-telangiectasia protein-deficient mice

Abstract

We have generated a mouse model for ataxia-telangiectasia by using gene targeting to generate mice that do not express the Atm protein. Atm-deficient mice are retarded in growth, do not produce mature sperm, and exhibit severe defects in T cell maturation while going on to develop thymomas. Atm-deficient fibroblasts grow poorly in culture and display a high level of double-stranded chromosome breaks. Atm-deficient thymocytes undergo spontaneous apoptosis in vitro significantly more than controls. Atm-deficient mice then exhibit many of the same symptoms found in ataxia-telangiectasia patients and in cells derived from them. Furthermore, we demonstrate that the Atm protein exists as two discrete molecular species, and that loss of one or of both of these can lead to the development of the disease.

Figure 1

(A) Schematic drawing of the Atm protein based on ref. . Shown are the leucine zipper (LZ) and PI-3 kinase (PI3K) domains. An arrow marks the approximate location where the mutation described in the work was introduced. (B) Schematic drawing of the wild-type atm gene (WT), the targeting construct (V), and the mutated atm allele (KO). RI, EcoRI; B, BamHI. Note that the Neo and atm genes are transcribed in opposite orientations. Use of the internal probe A in conjunction with EcoRI digestion in DNA blot analysis results in bands of 6.5 kb and 8.3 kb for the wild-type and mutated alleles, respectively.

Figure 2

(A) Blot analysis of tail DNA prepared from wild type (+/+), Atm heterozygous (+/−), and homozygous (−/−) mutant mice. DNA was cut with EcoRI and probed with probe A (Fig. 1B). (B) Blot analysis of poly(A)⁺ selected RNA from thymi of wild-type (WT) and Atm-deficient (KO) mice. The blot was probed with a mouse Atm cDNA fragment as described. Each lane contained 2 μg of RNA. Equal loading was verified by ethidium bromide staining (not shown). (C) Blot analysis of proteins extracted from fibroblasts derived from healthy (C) or AT (ATM) humans and from thymi (Thy.) and spleens (Spln.) of wild-type and Atm-mutant mice.

Figure 3

Atm-deficient mice are smaller than control littermates. The weight of male littermate mice of the indicated genotypes is shown as a function of time.

Figure 4

Cross section through a seminiferous tubule of a wild-type (A) and an Atm-deficient (B) adult mouse. Note that the developing sperm present in A is replaced by debris in B. (×200.)

Figure 5

DNA laddering in thymocytes of wild-type and Atm-deficient mice. Freshly isolated thymocytes were plated after either no irradiation (−) or irradiation at 3.5 Gy (+). DNA was prepared either immediately afterward (0) or after 8 hr incubation (8), and 10 μg was electrophoresed through a 1% agarose gel.