Down-regulation of CK2α correlates with decreased expression levels of DNA replication minichromosome maintenance protein complex (MCM) genes.

Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α') and two regulatory subunits (CK2ß). It is implicated in every stage of the cell cycle and in the regulation of various intracellular pathways associated with health and disease states. The catalytic subunits have similar biochemical activity, however, their functions may differ significantly in cells and in vivo. In this regard, homozygous deletion of CK2α leads to embryonic lethality in mid-gestation potentially due to severely impaired cell proliferation. To determine the CK2α-dependent molecular mechanisms that control cell proliferation, we established a myoblast-derived cell line with inducible silencing of CK2α and carried out a comprehensive RNA-Seq analysis of gene expression. We report evidence that CK2α depletion causes delayed cell cycle progression through the S-phase and defective response to replication stress. Differential gene expression analysis revealed that the down-regulated genes were enriched in pathways implicated in cell cycle regulation, DNA replication and DNA damage repair. Interestingly, the genes coding for the minichromosome maintenance proteins (MCMs), which constitute the core of the replication origin recognition complex, were among the most significantly down-regulated genes. These findings were validated in cells and whole mouse embryos. Taken together, our study provides new evidence for a critical role of protein kinase CK2 in controlling DNA replication initiation and the expression levels of replicative DNA helicases, which ensure maintenance of proliferative potential and genome integrity in eukaryotic cells.

Comparative Analysis of Non-viral Transfection Methods in Mouse Embryonic Fibroblast Cells.

Mouse embryonic fibroblast (MEF) cells are an important in vitro model for developmental biology, disease, and reprogramming studies. However, as with other primary cells, they are challenging to transfect. Although viral gene-delivery methods achieve high gene-delivery efficiency, challenges with cell mutagenesis and safety among others have led to the use and improvement of non-viral gene-delivery methods in MEF cells. Despite the importance of gene delivery in MEF cells, there is limited comparison of method/reagent efficacy. In this study, we compared the effectiveness of different gene-delivery methods and several reagents currently available in MEF cells by introducing a plasmid containing enhanced green fluorescent protein (EGFP). We analyze transfection efficiency by EGFP fluorescence. Our results suggest that two gene-delivery methods-electroporation and magnetofection in combination with a lipid reagent, are the most efficient transfection methods in MEF cells. This study provides a foundation for the selection of transfection methods or reagents when using MEF cells.

CK2α is essential for embryonic morphogenesis.

CK2 is a highly conserved serine-threonine kinase involved in biological processes such as embryonic development, circadian rhythms, inflammation, and cancer. Biochemical experiments have implicated CK2 in the control of several cellular processes and in the regulation of signal transduction pathways. Our laboratory is interested in characterizing the cellular, signaling, and molecular mechanisms regulated by CK2 during early embryonic development. For this purpose, animal models, including mice deficient in CK2 genes, are indispensable tools. Using CK2α gene-deficient mice, we have recently shown that CK2α is a critical regulator of mid-gestational morphogenetic processes, as CK2α deficiency results in defects in heart, brain, pharyngeal arch, tail bud, limb bud, and somite formation. Morphogenetic processes depend upon the precise coordination of essential cellular processes in which CK2 has been implicated, such as proliferation and survival. Here, we summarize the overall phenotype found in CK2α (-/- ) mice and describe our initial analysis aimed to identify the cellular processes affected in CK2α mutants.

Dynamic expression of a LEF-EGFP Wnt reporter in mouse development and cancer.

We have characterized a transgenic mouse line in which enhanced green fluorescent protein (EGFP) is expressed under the control of multimerized LEF-1 responsive elements. In embryos, EGFP was detected in known sites of Wnt activation, including the primitive streak, mesoderm, neural tube, somites, heart, limb buds, mammary placodes, and whisker follicles. In vitro cultured transgenic embryonic fibroblasts upregulated EGFP expression in response to activation of Wnt signaling by GSK3beta inhibition. Mammary tumor cell lines derived from female LEF-EGFP transgenic mice treated with the carcinogen 7, 12-dimethylbenz[a]anthracene (DMBA) also express EGFP. Thus, this transgenic line is useful for ex vivo and in vitro studies of Wnt signaling in development and cancer.