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Pharmacology |
Carrero-Feliciano, Marķa, Pontifical Catholic University of Puerto Rico; Lin, Lin; Shing-Shew, Shey* Department of Pharmacology and Physiology, Univ. of Rochester
Construction of recombinant adenovirus encoding mitochondrial ratiometric-pericam (r-pericam-mt)
Mitochondria are
the organelles that produce more than 90% of the energy of our body
by oxidative phosphorylation. Also mitochondria is involved in the
modulation of calcium levels in the cells. Certain diseases like
respiratory complications and cardiac diseases are related to
mitochondrial dysfunction. The calcium levels in the organelles are
very difficult to measure due to the possible signal contamination
from cytosol. A group of researchers made a chimeric protein named
Ratiometric-pericam-mitochondrial (R-pericam-Mt) that is able to
measure the mitochondrial calcium concentrations by changes in
fluorescence and spectral properties. The goal of our lab was to
create an adenovirus to express this chimeric protein and measure
the free mitochondrial [Ca2+] in cells which are
difficult to transfect with conventional transfection methods. The
gene encoding R-pericam-mt was first cloned into pShuttle-CMV
vector, which is the adenovirus transfer vector, to obtain R-pericam-mt-pShuttle-CMV.
The resulting plasmid was first tested by restriction enzyme
digestion. Transient transfection of this plasmid to QBI293-A cells
also confirmed the protein expression of R-pericam-mt by two
methods: fluorescence microscopy and western blot. The homologous
recombination in bacteria to produce adenovirus plasmid containing
pericam was performed by co-transformation of linearized R-pericam-mt-pShuttle-CMV
and pAdEasy-1 into BJ5183 cells. The restriction enzyme digestion
confirmed the recombination of these two plasmid between the right
arms and the origins of replication. After amplification of the
recombinant adenovirus plasmid, it was digested with Pac I and then
transfected into QBI293-A cells to produce virus and form viral
plaque.