Western blot analysis of CK2α and CK2β. ES cell extracts (A) and brain tissue extracts from adult mice (B) with different genotypes as indicated above the lanes were analyzed for CK2α and CK2β expression. For CK2β detection, the same result was obtained with two different antibodies. To ensure equal amounts of loading, an anti-actin antibody was used.

Failure of CK2β^−/− morula outgrowth in vitro. (A to D) Typical outgrowth of CK2β^+/+ and CK2β^+/− morulae. (E to H) Typical outgrowth of CK2β^−/− morulae. Panels A and E show cells at the morula stage, and panels B and F show cells at the blastocyst stage. No difference between individual embryos was seen at the morula and blastocyst stages. Outgrowth after 4 (C and G) and 7 (D and H) days in culture are shown. In contrast to CK2β^+/+ and CK2β^+/− embryos (D), CK2β^−/− embryos (H) did not develop an ICM, and they display vacuolated trophoblastic cells (TG). (I) Genotype determination of cells at the end of the blastocyst outgrowth experiment by PCR. Primers 1 and 4 detected the CK2β⁻ allele. Primers 5 and 4 amplified a 246-bp fragment from the CK2β wild-type allele only, not from the CK2β knockout allele, since the primer 5 binding site was lost after Cre deletion. Primers 6 and 4 amplified a 162-bp fragment from both the CK2β wild-type and knockout alleles (see Fig. 1A for locations of the primers), i.e., the absence of the 246-kb fragment indicates the absence of the CK2β wild-type allele.

Classical and conditional knockout of CK2β. (A) Gene-targeting strategy and deletion events after Cre expression. Maps of the CK2β wild-type allele, CK2β⁺, the targeting vector, the CK2β^3lox allele after gene targeting, and the resulting CK2β⁻ and CK2β^2lox alleles after Cre expression. Rectangles represent the seven exons of the CK2β gene. The area of the CK2β⁺ genomic region used for gene targeting is indicated by the thick horizontal line. loxP sites are indicated by black arrowheads. The positions of the 5′ and 3′ external probes and the internal probe used for Southern blot hybridization and the fragments detected for the different alleles are indicated above the CK2β⁺ allele and below the CKβ^2lox allele by black bars. The positions of primers (numbered 1 to 6) for PCR analysis are indicated by arrows below the CK2β⁺, CK2β^3lox, CK2β⁻, and CK2β^2lox alleles. Restriction enzyme cutting sites relevant for the detection of the different alleles are marked as follows: H, HindIII; E, EcoRI; and Xh, XhoI. (B) Identification of CK2β^3lox/+ ES cell clones. The correct targeting event was confirmed by the appearances of a 3.0-kb HindIII fragment after hybridization with the 5′ probe and of a 7.3-kb EcoRI fragment after hybridization with the 3′ probe, whereas the wild-type allele gave rise to 5.1- and 5.7-kb bands, respectively. The genotypes are indicated above the lanes. (C) Strategy to discriminate CK2β alleles by Southern blot hybridization (left) and PCR analysis (right). For Southern blot hybridization, genomic DNA was digested with XhoI and hybridized with the internal probe, which revealed fragments of specific sizes for the four different alleles. For PCR analysis, two of the four primers (designated 1 to 4) were used in three different combinations, as indicated below the lanes. Fragments of different sizes, depending on the alleles present, were obtained. The genotypes are indicated above the lanes. In the case of the PCR with primers 1 and 4 and the CK2β^+/− genotype, competition between the 2.6- and 0.3-kb products from the CK2β⁺ and CK2β⁻ alleles, respectively, led to amplification of only the small fragment specific for the CK2β⁻ allele. Figure 4 shows the results of PCR with primers 5 and 6.

Knockout of CK2β results in postimplantation lethality. (A to D) Embryos with normal morphology. (E to H) Embryos with abnormal morphology. Shown are dissected E7.5 embryos (A and E), BrdU labeling and hematoxylin staining of saggital sections of E7.5 (B and F) and E6.5 (C and G) embryos, and Hoechst staining of E6.5 embryos (D and H). In normal embryos at E7.5 (B), the amniotic (ac), exocoelomic (ecc), and extraplacental (epc) cavities can be seen; at E6.5 (C), the preamniotic canal (pc) is visible (20). In abnormal embryos at E6.5 (G), development is stopped: the embryos are smaller, and no preamniotic canal, only an ICM with a small, probably blastocoelic, cavity, can be seen.

Cre expression in CK2β^2lox/+ and CK2β^2lox/− ES cells. (A) Genotype determination of CK2β^2lox/+ and CK2β^2lox/− ES cells by PCR with primers 3 and 4 and 1 and 4 (Fig. 1A). (B) CK2β^2lox/+ and CK2β^2lox/− ES cells were infected with Cre-expressing retrovirus cultured for 4, 6, or 9 days as indicated and stained. Puromycin selection (puro) was applied as indicated. (C) Genotype determination, by PCR, of the CK2β^2lox/+ ES cells shown in panel B. In the infected CK2β^2lox/+ ES cells, the CK2β⁻ allele (−) could be detected 4 days after infection. Nine days after infection, the CK2β^2lox allele (2lox) had almost disappeared. Data shown are representative of four independent experiments.