Calmodulin (CaM) was shown to be essential for survival of
lower eukaryotes by gene deletion experiments. So far, no CaM
gene deletion was reported in higher eukaryotes. In vertebrates,
CaM is expressed from several genes, which encode an identical
protein, making it difficult to generate a model system to study
the effect ofCaMgene deletion. Here, we present a novel genetic
system based on the chicken DT40 cell line, in which the two
functional CaM genes were deleted and one allele replaced with
a CaM transgene that can be artificially regulated.Weshow that
CaM is essential for survival of vertebrate cells as they die in the
absence of CaM expression. Reversal of CaM repression or
ectopic expression of HA-tagged CaM rescued the cells. Cells
exclusively expressing HA-CaM with impaired individual calcium
binding domains as well as HA-CaM lacking the ability to
be phosphorylated at residues Tyr99/Tyr138 or trimethylated at
Lys115 survived and grew well. CaM mutated at both Ca2 binding
sites 3 and 4 as well as at both sites 1 and 2, but to a lesser
degree, showed decreased ability to support cell growth. Cells
expressing CaM with all calcium binding sites impaired died
with kinetics similar to that of cells expressing no CaM. This
system offers a unique opportunity to analyze CaM structurefunction
relationships in vivo without the use of pharmacological
inhibitors and to analyze the function of wild type and
mutated CaM in modulating the activity of different target systems
without interference of endogenous CaM.