Figure 1. Generation and characterization of Seckel Terc compound mice. (A) Outline of the ATR+/S/Terc+/ × ATR+/S/G2 Terc/ cross. G3 progeny inherit one set of short telomeres (from the G2 Terc/ parent) and one set of long telomeres (from the Terc+/ parent), but only G3 Terc+/* progeny will be telomerase proficient. (B) The Seckel mutation does not affect telomerase activity. The TRAP assay shows successful telomerase reconstitution in G3 Terc+/* MEFs compared with their G3 Terc/* littermates. Protein concentrations used are indicated above each lane. As a negative control, extracts were treated with RNase (+) or left untreated (). IC, internal control. (C) Reduced ATR expression in ATRS/S cells. Western blot showing ATR expression levels in ATR+/+ and ATRS/S MEFs. Actin loading control is indicated below. (Brown and Baltimore, 2000; de Klein et al., 2000). Thus, the effects of ATR depletion in adult mice and cells have been tested only in the context of in vitro cell culture models or conditional ATR deletion in already adult animals (Ruzankina et al., 2007). To address the role of ATR in the regulation of telomerase recruitment, we used the Seckel syndrome mouse model recently generated by us (Murga et al., 2009). In a sub- set of human patients with Seckel syndrome, a single point mutation in ATR exon (E) 9 leads to aberrant splicing and se- verely decreased ATR protein expression and is the cause of growth retardation, microcephaly, brain and skeletal abnor- malities, and highly unstable cellular genomes (O’Driscoll et al., 2003; Casper et al., 2004). The Seckel mouse model used in this study harbors a humanized ATR allele in which the region encompassing murine E8, E9, and E10 has been swapped by the human counterpart containing the Seckel mutation in E9 (Murga et al., 2009). This leads to a splicing defect in the E8–E10 boundary, which severely reduces ATR levels and re- capitulates the human Seckel syndrome phenotypes in mice (Murga et al., 2009). Mice lacking the RNA component of telomerase, Terc, show progressive telomere shortening from the first genera- tion until the third or fourth generation (when in a pure C57BL6 background), by which stage they present critically short telomeres, defective stem cell proliferative capacity, in- fertility as the result of germ cell apoptosis, and increased ge- nomic instability (Blasco et al., 1997; Herrera et al., 1999; Flores et al., 2005). We and others have shown that the restora- tion of telomerase activity in these mice, by reintroduction of one copy of the Terc gene, rescues critically short telomeres and reverses chromosomal instability and cell and tissue de- fects associated with late-generation telomerase deficiency (Hemann et al., 2001; Samper et al., 2001a; Siegl-Cachedenier et al., 2007). To directly test whether ATR proficiency is necessary for telomerase action specifically on short telomeres in primary mouse embryonic fibroblasts (MEFs), we crossed ATR+/S/ Terc+/ females with G2 ATR+/S/Terc/ males to generate litter- mate populations of ATRS/S and ATR+/+ mice that were also either G3 Terc/ or G3 Terc+/. The G3 progeny of these crosses inher- ited a set of chromosomes with short telomeres from the male G2 ATR+/S/Terc/ parent and a set of chromosomes with nor- mal telomeres from the female ATR+/S/Terc+/ parent (and are hereafter referred to as G3 Terc/* or G3 Terc+/*; Fig. 1 A), but only the G3 Terc+/* progeny would inherit a copy of the Terc gene and be telomerase proficient (Fig. 1 B). To confirm ATR abrogation in MEFs derived from the ATRS/S progeny, we first demonstrated that ATR expression is undetectable in ATRS/S compared with ATR+/+ MEFs, as determined by Western blot- ting experiments (Fig. 1 C). As expected, Terc deficiency did not affect ATR expression levels (Fig. 1 C, compare the first and second lanes with the third and fourth lanes). We also confirmed that reintroduction of the Terc allele successfully reconstituted telomerase activity in both ATR+/+/G3 Terc+/* and ATRS/S/G3 Terc+/* MEFs (Fig. 1 B), demonstrating that telomerase processivity and activity is not affected by ATR depletion, which is similar to what has been previously reported for cells lacking ATM (Sprung et al., 1997). Telomere defects in ATR-deficient mice • McNees et al. 641