© 1999 Macmillan Magazines Ltdletters to nature16 8 NATURE|VOL 397|14 JANUARY 1999|www.nature.complated in triplicate into 12-well plates. At various time points cells were stainedwith crystal violet (Sigma) and the optical density at 590 nm was determined15.Values were normalized to the optical density at day 0 (20 h after plating).Phoenix producer cells were used to generate retroviral stocks as described15.For infection, subcon¯uent passage 1 MEF cultures were incubated at 37 8Cwith viral supernatant in the presence of 4 mgml-1polybrene (Sigma). After 6 hthe viral supernatant was diluted 1:3 with complete medium and left on thecells for 42 h. Appropriate dilutions of viral supernatants were used such that100% of MEFs were infected.BrdU-incorporation assay and western blotting. MEFs were grown oncoverslips and incubated for 4 h with 10 mM BrdU (Amersham). Cells werewashed in PBS, ®xed for 15 min at -20 8C in 5% acetic acid/95% ethanol andincubated in PBS. Fixed cells were incubated for 20 min in 2 M HCl/0.5%Triton-X100, for 30 min in blocking solution (5% fetal calf serum, 5% normalgoat serum in PBS/0.02% Triton-X100), for 1 h with 1:10 diluted anti-BrdUmonoclonal antibody (DAKO) in blocking solution, and overnight at 4 8C with1:50 diluted ¯uorescein isothiocyanate (FITC)-conjugated goat anti-mouseantibody (Jackson Immuno Research Labs) and 4,-6-diamidino-2-phenylin-dole (DAPI) in blocking solution; this was followed by 3 washes for 5 min eachin PBS/0.02% Triton-X100. Cells were embedded in Vectastain (Vector Labs)and the percentage of BrdU-labelled cells (FITC:DAPI ratio) was quanti®edusing a ¯uorescence microscope. For protein analysis, cells were washed withPBS, scraped and lysed on ice in RIPA buffer (150 mM NaCl, 1% NP40, 0.1%SDS, 0.5% DOC, 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, pH 8.0, 0.2 mMphenylmethylsulphonyl¯uoride (PMSF), 0.5 mM dithiothreitol (DTT)).Cleared lysates were assayed for protein concentration. Equal amounts ofprotein were separated on 12.5% SDS±PAGE and transferred to nitrocellulose.Western blot analysis was according to standard methods using enhancedchemiluminescence (Amersham). A list of antisera used is available on request.Expression analysis. Total RNA was extracted using guanidinium thiocya-nate, separated on 1.2% agarose, transferred to nitrocellulose and hybridizedaccording to standard procedures with32P-labelled probes speci®c for exon 1aof mouse p16, for exon 1b of mouse p19Arf, for mouse p15 or for ratglyceraldehyde-3-phosphate dehydrogenase (GAPDH). For semiquantitativereverse transcription with polymerase chain reaction (RT-PCR), ®rst-strandcomplementary DNA was generated from 1 mg total RNA using Superscript IIRT (Gibco) and oligo-dT primer according to the manufacturer's instructions.Primer sequences are available upon request. PCR reactions were performed on®ve-times serial dilutions of ®rst-strand cDNA in 50 ml containing 1´ Taq PCRbuffer, 1.5 mM MgCl2, 200 mM dNTPs, 0.5 mM of each of four primers, 1 mlof®rst-strand cDNA template and 1.25 units of Taq DNA Polymerase (Gibco),using 35 cycles of denaturation (94 8C, 1 min), annealing (60 8C, 45 s) andextension (72 8C, 2 min). Products were resolved on 2% NuSieve agarose gels.Generation of bmi-1-/-ink4a-/-mice. bmi-1+/-FVB mice4and ink4a+/-miceon a mixed 129/Sv; C57BL/6 genetic background24were crossed to generatebmi-1+/-ink4a+/-mice, which were subsequently intercrossed to generatedouble-knockout offspring together with control littermates. Mice weregenotyped routinely by PCR or Southern blot analysis of tail DNA.Cell count and ¯ow-cytometric analysis. Cell suspensions of lymphoidorgans were prepared by mincing the tissue though an open ®lter chamber. Cellsuspensions were depleted of erythrocytes and the number of nucleated cellswas determined with a Casy-1 TT automated cell counter (SchaÈfe, Reutlingen,Germany). Flow cytometry with standard B- and T-cell differentiation wasdone nearly as described10.Histological analysis. Brains were ®xed in 4% formaldehyde in PBS, paraf®n-embedded and cut into 4-mm serial sagittal sections. Sections at different levelswere stained with haematoxylin and eosin.Received 10 September; accepted 12 November 1998.1. van Lohuizen, M. et al. Identi®cation of cooperating oncogenes in Em-myc transgenic mice byprovirus tagging. Cell 65, 735±752 (1991).2. Haupt, Y., Alexander, W. S., Barri, G., Klinken, S. P. & Adams, J. M. Novel zinc ®nger gene implicatedas myc collaborator by retrovirally accelerated lymphomagenesis in Em-myc transgenic mice. Cell 65,753±763 (1991).3. van Lohuizen, M., Frasch, M., Wientjens, E. & Berns, A. Sequence similarity between the mammalianBmi-1 proto-oncogene and the Drosophila regulatory genes Psc and Su(z)2. Nature 353, 353±355 (1991).4. van der Lugt, N. M. T. et al. Posterior transformation, neurological abnormalities, and severehematopoietic defects in mice with a targeted deletion of the Bmi-1 proto-oncogene. Genes Dev. 8,757±769 (1994).5. Alkema, M. J. et al. MPc2, a new murine homologue of the Drosophila Polycomb protein is a memberof the mouse Polycomb transcriptional repressor complex. J. Mol. Biol. 273, 993±1003 (1997).6. Alkema, M. J., van der Lugt, N. M. T., Bobeldijk, R. C., Berns, A. & van Lohuizen, M. Transformationof axial skeleton due to overexpression of Bmi-1 in transgenic mice. Nature 374, 724±727 (1995).7. Paro, R. Propagating memory of transcriptional states. Trends Genet. 11, 295±298 (1995).8. van Lohuizen, M. Functional analysis of mouse Polycomb-group genes. Cell. Mol. Life Sci. 54, 71±79(1998).9. Gould, A. Functions of mammalian Polycomb group and trithorax group related genes. Curr. Opin.Genet. Dev. 7, 488±494 (1997).10. Alkema, M. J., Jacobs, H., van Lohuizen, M. & Berns, A. Perturbation of B and T cell development andpredisposition to lymphomagenesis in Em-Bmi1 transgenic mice require the Bmi1 RING ®nger.Oncogene 15, 899±910 (1997).11. Dimri, G. P. et al. A biomarker that identi®es senescent human cells in culture and aging skin in vivo.Proc. Natl Acad. Sci. USA 92, 9363±9367 (1995).12. Hara, E. et al. Regulation of p16 CDKN2 expression and its implications for cell immortalisation andsenescence. Mol. Cell. Biol. 16, 859±867 (1996).13. Alcorta, D. A. et al. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicativesenescence of normal human ®broblasts. Proc. Natl Acad. Sci. USA 93, 13742±13747 (1996).14. Zindy, F., Quelle, D. E., Roussel, M. F. & Sherr, C.