Securin is not required for cellular viability, but is required for normal growth of mouse embryonic fibroblasts Junjie Mei, Xingxu Huang, Pumin Zhang

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Securin is not required for cellular viability, but is required for normal growth of mouse embryonic fibroblasts Junjie Mei, Xingxu Huang, Pumin Zhang Current Biology Volume 11, Issue 15, Pages 1197-1201 (August 2001) DOI: 10.1016/S0960-9822(01)00325-6

Figure 1 The targeted disruption of securin in mouse ES cells. (a) A diagram of the securin disruption strategy. Two kinds of targeting vectors are shown, one utilizing PGK-Neo as a positive selection marker, and the other utilizing PGK-HPRT. Both vectors delete exons 1 and 2 of securin. pMC1-TK is placed at the end of homology for negative selection. KnR is the kanamycin resistance gene left over from the construction of the PGK-Neo targeting vector via homologous recombination in E. coli. Probes (a and b) for Southern analysis are indicated. (b) Southern blot analysis of genomic DNA isolated from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant ES cells. Restriction enzymes used to digest the DNA are indicated at the bottom of each blot. (c) Northern blot analysis of securin mRNA levels in total RNA isolated from wild-type, heterozygous, and homozygous mutant ES cells. The same blot was reprobed with GAPDH for a loading control. (d) RT-PCR analysis of total RNA isolated from the tail tissues of wild-type (+/+) and securin mutant (−/−) mice, with a forward primer located in exon 3 and a reverse primer located in exon 4 Current Biology 2001 11, 1197-1201DOI: (10.1016/S0960-9822(01)00325-6)

Figure 2 The response of ES cells to colcemid treatment. (a) Cell cycle analysis. Wild-type and securin mutant ES cells, asynchronously growing or treated with colcemid for 6, 12, and 24 hr, were analyzed with flow cytometry. The percentage of each cell cycle phase is indicated. (b) Mitotic indices. The results are from three independent experiments Current Biology 2001 11, 1197-1201DOI: (10.1016/S0960-9822(01)00325-6)

Figure 3 The chromosome spread of wild-type and mutant ES cells. (a) Normal metaphase chromosomes from wild-type unsynchronized ES cells. (b) A portion of chromosome spread from wild-type ES cells demonstrating sister separation. (c) The chromosome spread from 24-hr colcemid-treated wild-type ES cells. (d) The chromosome spread from 24-hr colcemid-treated securin−/− ES cells. The arrows indicate cohesion in the centromere region. The scale bar indicates 50 μm Current Biology 2001 11, 1197-1201DOI: (10.1016/S0960-9822(01)00325-6)

Figure 4 Phenotypic analyses of securin-null fibroblasts. (a) A growth curve assay of wild-type, securin heterozygous, and securin-null mouse embryonic fibroblasts. (b) A cell cycle profile of asynchronously growing and colcemid-treated wild-type and securin-null fibroblasts. The percentage of each cell cycle phase is indicated. (c) A diagram showing the percentages of metaphase (Meta) versus anaphase (Ana) of mitotic cells in asynchronously growing wild-type and securin mutant fibroblasts Current Biology 2001 11, 1197-1201DOI: (10.1016/S0960-9822(01)00325-6)