Nucleic Acids Research, 2010, Vol. 38, No. 11 3537 Figure 1. Generation of histone H1-null DT40 cells (A) Flow for generating histone H1 mutant DT40 cells. (B) Schematic diagram of the homologous recombination for 02H1 gene disruption. Targeting vectors (pD02H1/hyg and pD02H1/bleo) and the wild-type and targeted alleles are indicated with the expected sizes of HindIII-digested fragments. 02H1 and other histone genes are indicated as closed and open arrows, respectively. A bar represents the position of the 02H1-30 probe for Southern hybridization. (C) Southern blot analysis. Genomic DNA was prepared from wild-type, H1 10/12, H1 11/12, H1 11/12+ﬂox, H1 12/12+ﬂox and H1 12/12 #1 and #2 cells, digested with HindIII, and analyzed by Southern blotting using the 02H1-30I probe (upper panel) and the eGFP probe (lower panel). (D, E) Histone H1 proteins are not detected in the HH11-n1u1/l1l2m,Hut1an1t1/c1e2+llsﬂ.oxH, 2HSO14-12a/1n2d+ﬂHox2SaOnd4/HHC1lO124/-1e2xt#r1acatns,dw#h2icchelclso,natanidn saelplahriasttoednebsyan14d%H1S,DrSes-PpeAcGtivEe.lyG,ewlserweeprreepstaariendedfrwomithwCilBd-Bty(pDe,) Hor1 tr1a0n/1s2-, ferred to a membrane for blotting with anti-GFP to detect H1R-eGFP (E). Asterisks in (D) indicate probable HMG proteins. A representative experiment is shown in all ﬁgures. Global chromatin structure and cell cycle progression are maintained in H1-null DT40 cells We then examined chromatin structure in histone H1-null mutant cell lines during interphase and mitosis by staining with DAPI. It has been reported that chromosomes are condensed normally in mitotic Xenopus egg extract from which H1 has been immunodepleted, whereas extended chromosomes associated with misalignment and missegregation were found in H1-depleted repli- cated chromatin in diﬀerent Xenopus extracts (26,27). Confocal microscopy analysis revealed that there were no clear morphological diﬀerences between wild-type and H1-null (H1 12/12) cells, either in condensed mitotic chromosomes (Figure 4A) or in interphase nuclei (Figure 4B). Furthermore, in investigating many paraformaldehyde-ﬁxed mitotic cells and metaphase spreads prepared for chromosome breakage analysis (see below), we found no drastically extended ﬁbers as reported in H1-depleted Xenopus cycling extracts (27). Since H1-null (H1 12/12) and most deﬁcient (H1 11/12) DT40 cells showed growth defects, we performed a cell cycle analysis by measuring BrdU uptake and DNA content. The distribution of G1, S and G2/M populations was mostly similar between wild-type and H1 12/12 cells (Figure 4C). We also monitored the accumulation of cells positive for H3S10ph (an M-phase marker) in the culture after the addition of colcemid (Figure 4D, right). The ratio of H3S10ph-positive cells in steady state (0 h after- colcemid addition) and the accumulation kinetics did