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. 2001 Dec 11;98(26):15084–15088. doi: 10.1073/pnas.261574698

Figure 1.

Figure 1

DNA-PKcs-deficient MEFs exhibit similar telomere lengths and telomerase activities when compared with wild-type MEFs. (A) Southern analysis of MEFs. Early passage MEFs from independently isolated littermates were prepared from wild-type (lanes 7 and 8), DNA-PKcs+/− (lanes 1, 2, 5, and 6), and DNA-PKcs−/− (lanes 3 and 4). Gel plugs containing genomic DNAs were digested with RsaI and HinfI (odd number lanes) or undigested (even number lanes), fractionated by pulse-field gel electrophoresis, and hybridized with the telomeric specific [TTAGGG]3 probe. The approximate sizes of the products (kb) are indicated based on molecular weight markers. The Southern hybridization signal observed with the [TTAGGG]3 probe under these conditions was sensitive to BAL-31 exonuclease digestion, suggesting that this is telomeric DNA (data not shown). (B) PCR analysis for genotyping. Using the specific primer pairs (see Materials and Methods), wild-type and targeted alleles were amplified as products of 450 bp and 360 bp, respectively. Lanes 2 and 4 show the DNA-PKcs+/− pattern, lane 3 shows the DNA-PKcs−/− pattern, and lane 5 shows the wild-type pattern. Lane 1 contains a size marker. (C) Telomerase activity in DNA-PKcs-deficient MEFs. TRAP assay was performed after 30 PCR cycles on cell extracts (10, 102, and 103 cells) prepared from DNA-PKcs−/− (lanes 1–3), DNA-PKcs+/− (lanes 4–6), and wild-type (lanes 7–9) MEFs. In lanes 10–12, a serial dilution of HeLa cell lysate was run as a positive control for quantitating relative telomerase activity levels. Lane 13 contains a negative control without cell lysate. IC denotes a standard internal control for PCR efficiency.