ASSESSMENT OF TELOMERE LENGTH IN HEMATOPOIETIC INTERPHASE CELLS USINGIN-SITU HYBRIDIZATION AND DIGITAL FLUORESCENCE MICROSCOPY

Telomeres are G/C-rich repetitive DNA sequences at the end of all euka ryotic chromosomes. The loss of telomeric repeat sequences during cell divisions has been proposed as a possible mechanism for cell senescen ce. The standard procedure for measurement of telomere length is South ern blot (SB) hybridization with a telomere-specific probe. However, i n using this technique no information can be obtained on variation in telomeric fragments due to interchromosomal, intrachromosomal, and int ercellular differences. Lansdorp et al. (Hum Mol Genet 5:685-691, 1996 ) developed a method to measure individual telomeres, using in situ hy bridization on metaphase chromosomes, employing peptide nucleic acid ( PNA) probes and digital fluorescence microscopy. In this paper we desc ribe a method that can be used to assess telomeric length in interphas e cells. An algorithm was developed to measure the total intranuclear fluorescence in situ hybridization (FISH) signal, which features accur ate correction for the local autofluorescence. Application of this met hodology to samples of fetal liver, umbilical cord blood, and adult bo ne marrow cells showed a gradual decrease of average telomeric length. Southern blot analysis and PNA FISH measurements on chromosomes in th e same samples showed similar results. Advantages of interphase measur ements include the possibility of studying nonproliferating cells, thu s avoiding selection and cell culturing. (C) 1998 Wiley-Liss, Inc.