Original Contributions Influences of inbreeding and genetics on telomere length in mice Erin L. Manning, 1 Janet Crossland, 2 Michael J. Dewey, 2 Gary Van Zant 1 1 Departments of Physiology and Internal Medicine, Division of Hematology/Oncology, Markey Cancer Center, Room CC-408, University of Kentucky, 800 Rose St., Lexington, Kentucky 40536-0093, USA 2 Department of Biological Sciences and Peromyscus Stock Center, University of South Carolina, Columbia, South Carolina 29208, USA Received: 12 December 2000 / Accepted: 9 January 2002 Abstract. We measured telomere lengths of blood leukocytes in several inbred and outbred mammalian species, using a telomere- specific fluorescent probe and flow cytometry. Humans, non- human primates, and three outbred populations of Peromyscus mice (Peromyscus leucopus, Peromyscus maniculatus, and Pero- myscus polionotus) have short telomeres. Two common strains of laboratory mice, C57BL/6J and DBA/2J, have telomeres several times longer than most other mammals surveyed. Moreover, the two inbred laboratory mouse strains display significantly different telomere lengths, suggesting the existence of strain-specific ge- netic determinants. To further examine the effects of inbreeding, we studied three Peromyscus leucopus inbred lines (GS109, GS16A1, and GS16B), all derived from the outbred P. leucopus stock. Telomeres of all three inbred lines are significantly length- ened relative to outbred P. leucopus, and the three lines display strain-specific significantly different telomere lengths, much like the C57BL/6J and DBA/2J strains of M. musculus. To further characterize the genetic inheritance of telomere length, we carried out several crosses to obtain hybrid F 1 mice between parental strains displaying the phenotype of long and short telomeres. In all F 1 mice assayed, peripheral blood leukocyte telomere length was intermediate to that of the parents. Additionally, we generated F 2 mice from a cross of the (P. leucopus outbred × GS16B)F 1 . Based on the distribution of telomere length in the F 2 population, we determined that more than five loci contribute to telomere length regulation in Peromyscus. We concluded that inbreeding, through unknown mechanisms, results in the elongation of telomeres, and that telomere length for a given species and/or sub-strain is ge- netically determined by multiple segregating loci. Telomeres, the oligonucleotide repetitive sequences that comprise the ends of eukaryotic chromosomes, are evolutionarily conserved structures that, in mammals, play roles in the replicative senes- cence of cells in vitro and in cancer progression in vivo (Blackburn 2000; Campisi et al. 2001; Sherr and DePinho 2000). In the ab- sence of telomerase, telomeres shorten with cell replication. Thus, it is thought that the stabilizing effects of telomeres on chromo- somes are lost in dividing cells, at least in vitro, as they erode to critically short lengths (Allsopp et al. 1992; Hackett et al. 2001; Harley et al. 1990). End-to-end chromosome fusions and genomic instability are among the genetic consequences. The relationship, if any, between telomere dynamics and organismal aging and lon- gevity is largely unknown. Cells of inbred laboratory mice, in which studies of aging and cancer are typically carried out, have anomalously extended telomeres that are several times longer than those of human cells (Hemann and Greider 2000; Kipling and Cooke 1990; Starling et al. 1990). Laboratory mice deficient in mTR, the RNA component of telomerase, a native enzyme that extends and thus stabilizes telomeres in the face of replicative stress, show few physiological consequences for several genera- tions, but eventually fail to reproduce owing to decreased fertility, as well as increased embryonic lethality due to a neural tube clo- sure defect (Herrera et al. 1999a). Late-generation, mTR-deficient mice show defects in highly proliferative tissues (Blasco et al. 1997; Lee et al. 1998). Moreover, telomerase knockout mice gen- erated on a genetic background characterized by strain-specific shorter telomeres show deleterious effects in earlier generations (Herrera et al. 1999b). The physiological effects of telomerase deficiency in humans are manifested in dyskeratosis congenita, a congenital disease caused by mutation of the RNA component of telomerase (Mitchell et al. 1999; Vulliamy et al. 2001). Since human telomeres are shorter than those of most mice, rapidly proliferating tissues are affected early in life in people suffering from the disease, and death usually results in early adulthood from bone marrow failure. However, recent findings also suggest that telomere dynamics are more complex than originally thought and that a cell’s mitotic history and telomerase expression status alone are not sufficient to describe a cell’s probability of senescence or neoplastic transfor- mation (Blackburn 2000). The regulation of the t-loop structure of the telomeric end is regulated by telomeric binding proteins, such as TRF2 in humans. The t-loop structure appears to play an im- portant role in protecting the 3' overhang from nuclease degrada- tion, and also in limiting the access of telomerase to the telomeric end (Blackburn 2000, 2001; Campisi et al. 2001; de Lange 2001). For mice, even reproductive strategies such as inbreeding, out- breeding, or domestication are thought to influence telomere length (Bickle et al. 1998; Hemann and Greider 2000). In this report, we further examined the effect of breeding strategy in another species, the white-footed mouse, Peromyscus leucopus, and two other congeneric species. We conclude that telomere length (a) is increased by inbreeding and (b) is genetically deter- mined by multiple segregating loci that establish a base telomere length upon which inbreeding builds its epistatic effect. Materials and methods Animals. Blood from all Peromyscus species was obtained from animals housed at the Peromyscus Stock Center, Columbia, S.C. Blood from non- inbred Mus musculus castaneus and Mus spretus mice housed at the Roswell Park Cancer Institute, Buffalo, N.Y., was provided by Rosemary Elliott. Blood from Rhesus monkeys (Texas Primate Center, Alice, Tex.) housed at the University of Kentucky was provided by Don M. Gash. All other mice were housed under barrier conditions in the University of Ken- tucky Division of Lab Animal Resources with food and water available ad libitum. DBA/2J, C57BL/6J, CAST/EiJ, SPRET/EIJ, and (C57BL/6J × DBA/2J)F 1 mice were obtained from The Jackson Laboratory, Bar Harbor, Me. Black Swiss, Swiss Webster, and ICR outbred mice were obtained from Taconic, Germantown, N.Y. Correspondence to: G. Van Zant; E-mail: gvzant1@uky.edu © Springer-Verlag New York Inc. 2002 Mammalian Genome 13, 234–238 (2002). DOI: 10.1007/s003350020027 Incorporating Mouse Genome