Targeted disruption of mouse centromere protein C gene leads to mitotic disarray and early embryo death - PubMed (original) (raw)

Targeted disruption of mouse centromere protein C gene leads to mitotic disarray and early embryo death

P Kalitsis et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Centromere protein C (CENPC) is a key protein that has been localized to the inner kinetochore plate of active mammalian centromeres. Using gene targeting techniques, we have disrupted the mouse Cenpc gene and shown that the gene is essential for normal mouse embryonic development. Heterozygous mice carrying one functional copy of the gene are healthy and fertile, whereas homozygous embryos fail to thrive. In these embryos, mitotic arrest and gross morphological degeneration become apparent as early as the morula stage of development. The degenerating embryos demonstrate highly irregular cell and nuclear morphologies, including the presence of a large number of micronuclei. Mitotic chromosomes of these embryos display a scattered and often highly condensed configuration and do not segregate in an ordered fashion. These results describing the phenotype of the mutant mouse embryos indicate that CENPC has a direct role in the mitotic progression from metaphase to anaphase.

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Figures

Figure 1

Figure 1

Targeted disruption of the mouse Cenpc gene. (A) Gene replacement constructs and restriction maps. (i) Mouse CENPC protein, showing the amino acid positions of the minimum centromere-targeting domain (18) and the location of this domain downstream of the gene-disruption site. (ii–iv) Restriction maps for the Cenpc locus covering exons 8 to 12 (ii), the gene replacement construct (iii), and the Cenpc locus after targeted disruption (iv). Black boxes represent exons. The selectable marker cassette contained in the targeting construct consists of a splice acceptor site (SA), a picornaviral internal ribosome-entry site (IRES), a _lacZ_–neomycin-resistance fusion gene (βgeo), and a simian virus 40 polyadenylation sequence (pA) (27). A 1.2-kb _Xba_I fragment (designated 3′ probe) located downstream of the targeted region was used in the Southern screening strategy and detected a 6.1-kb wild-type _Eco_RI fragment in the untargeted locus or a 6.8-kb _Eco_RI fragment in the targeted locus. Arrows indicate positions of primers used in mouse tail and embryo PCR. Crosses denote expected sites of homologous recombination. Abbreviations for restriction enzymes are E, _Eco_RI; S, _Sal_I; Xb, _Xba_I; and Xh, _Xho_I. (B) Southern blot analysis of wild-type and correctly targeted ES cell lines. The sizes of wild-type 6.1-kb and homologous recombinant 6.8-kb bands are shown on the right. (C) PCR genotyping of mouse tail DNA or postimplantation embryos. The primer set S-W gives a 995-bp wild-type Cenpc product, whereas the neomycin-Cenpc primer set, neo1-S, gives a 580-bp targeted product.

Figure 2

Figure 2

PCR analysis of preimplantation embryos from +/− × +/− crosses. (A) Nested PCR strategy for genotyping embryos up to the blastocyst stage. Cenpc primer pairs include AK–AL for first-round synthesis and AM–AN for second-round synthesis, giving a final product of 322 bp for the untargeted allele. β-geo primer pairs include GF1–GR1 for first-round synthesis and GF2–GR2 for second-round synthesis, giving a final product of 248 bp for the targeted allele. Abbreviations for restriction enzyme sites are (Xh) _Xho_I and (S) Sal_I. The black box indicates exon 8 of the mouse Cenpc gene (see Fig. 1_A). (B) Nested PCR genotyping of day-3.5 embryos.

Figure 3

Figure 3

Morphological analysis of Giemsa-stained day-3.5 embryos from +/− × +/− crosses. (A and B) Normal embryos, showing regular, round interphase nuclei and a number of cells undergoing mitosis. (C–F) Putative Cenpc homozygous mutant embryos, showing oversized interphase nuclei (blue arrows), scattered and highly condensed metaphase chromosomes (red arrows), and an abundance of micronuclei (black arrows). (×200.)

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