Telomere length is maintained by the ribonucleoprotein complex telomerase 
. However, telomerase expression in humans occurs primarily in early development, germ cells, and in stem cells and is not detected in primary cells 
. Most human tumor cells have detectable telomerase activity, however some proliferating tumors lack telomerase and thus maintain telomeres by alternative mechanisms that are collectively termed ALT for alternative lengthening of telomeres 
. While mTR−/− mice have a reduced frequency and rate of tumor formation, some tumors form and can grow rapidly in these mice 
. However, the mechanism by which tumors grow in the absence of telomerase is not known.
Telomerase deficient mice were initially generated by deleting the gene encoding the telomerase RNA (mTR) component 
. Although mTR−/− mice lack telomerase activity, no phenotype is observed in the first generation, due to the long telomeres observed in laboratory mouse strains 
. When mTR−/− mice are bred, progressive telomere shortening occurs in successive generations. Early generation, mTR−/−G1, mice are obtained by crossing mTR+/− mice. Crossing the knockouts through successive generations results in mTR−/− G2–G6 generations. Late generation mTR−/− G4–G6 mice have short telomeres and show loss of fertility due to germ cell apoptosis. Wild-derived mouse strains such as CAST/EiJ have significantly shorter telomere length distributions, similar to humans 
. CAST/EiJ mTR+/− mice bred for increasing generations show progressive telomere shortening and loss of tissue renewal capacity 
. The phenotypes in the mTR+/− CAST/EiJ mice mimic the human genetic disease, dyskeratosis congenita, due to haploinsuffiency for telomerase 
. Wildtype mice derived from an intercross between late generation heterozygous parents (termed WT*) have shorter telomeres and also display tissue renewal defects 
. Thus, telomere shortening and consequent loss of tissue renewal capacity occurs in CAST/EiJ mice even in the presence of telomerase, and provides the opportunity to examine the effects of short telomeres in the presence of telomerase.
Several lines of evidence indicate that ALT occurs by DNA recombination in human tumors and immortalized cells 
. First, the initial description of ALT demonstrated that the telomeres are exceptionally long and heterogeneous in human tumors and immortalized cell lines which lack telomerase 
. Second, telomere lengths in ALT cells fluctuate during proliferation, and this fluctuation can be detected by examining the change in the telomere lengths at the p- and q-arm of the Y-chromosome in a rapidly growing culture 
. Third, a unique plasmid sequence integrated as single copy at the telomere was found duplicated at other chromosomes following serial transfer of human ALT cells 
. ALT associated nuclear promyelocytic leukemia bodies (APBs) are found in a subset of human ALT cell lines and contain various recombination proteins 
. However it is uncertain what functional role APBs contribute to ALT mechanisms and they are often considered as only a marker for some ALT cell lines 
. Finally, human ALT tumors show frequent telomere sister chromatid exchanges (T-SCEs), that are detectable using chromosome orientation FISH (CO-FISH) 
Evidence that recombination contributes to telomere length maintenance was initially discovered in Saccharomyces cerevisiae
. Yeast lacking an essential component of telomerase showed progressive telomere shortening and loss of viability, however survivors appeared after successive streaking of the colonies 
. Studies of these yeast survivors showed that telomere recombination contributes to length maintenance, and requires the RAD52 pathway 
. Survivors can be classified as Type I or Type II based on their telomere patterns and growth rate 
. Type I survivors require Rad51, Rad54, Rad55 and Rad57 
. The telomeres of Type I survivors are short and the cells have amplified Y′ sequence. They are likely generated by Rad51-dependent break-induced replication (BIR). Type II survivors grow much more rapidly than Type I survivors. They have elongated telomere sequence tracts, require Rad59, Rad50 and other components of the MRX complex, and are predominately generated by Rad51-independent BIR 
. Therefore, in yeast, telomere elongation in the absence of telomerase occurs mostly though BIR 
. Studies in Kluyveromyces lactis
have also provided insight on telomere recombination mechanisms. In particular, K.lactis
deleted for telomerase (ter1
) showed that telomere recombination is initiated by short telomeres 
. In mouse and human cells the T-SCE assay, which is frequently used to measure telomere recombination, will not detect recombination by BIR mechanisms. Further, T-SCEs are exchanges and thus will not result in net telomere elongation as occurs in BIR. Thus we sought to use other assays to examine telomere recombination in telomerase null mouse cells.
To examine the role of short telomeres during telomere recombination in mammalian cells we assayed cells using pq-ratios, outliers, CO-FISH, and Q-FISH from two different strains of telomerase deficient (mTR−/−) mice. We found that late passage CAST/EiJ mouse embryonic fibroblasts (MEFs) and Eμmyc+mTR−/− lymphomas with short telomeres, exhibit telomere maintenance with minimal changes to the overall length distribution. Consistent with telomere recombination, we observed an increase in pq-ratio changes and outliers in mouse cells with increasing numbers of short telomeres. We directly showed that subtelomeric recombination is increased in cells with elevated pq-ratio changes. These pq-ratio changes were seen associated with short telomeres even in telomerase positive cells, suggesting that telomerase itself does not protect against recombination. Our data suggest that, several distinct recombination-based mechanisms can contribute to telomere maintenance in mammalian cells.