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Expression Analysis of a Potassium Channel Regulator and
Generation of the Knock-Out Construct
Jennifer Thompson , Tara St. Amand, Ken Chien. Institute of Molecular
Medicine, University of California, San Diego. La Jolla, Ca 92092
Introduction
The expression of KCR-1, a K+ channel regulator, within the conduction system
may play a pivotal role in normal electrophysiological functioning of the heart. Normal
electrophysiological functioning of the heart is mandatory in order to prevent cardiac
sudden death. Cardiac sudden death is caused by non-sequential or rapid electrical
impulses within the heart.1 This irregular heart rhythm (arrhythmia) causes the heart to
stop beating suddenly.
Normal electrophysiological function of the heart depends on proper development
of the cardiac conduction system. The conduction system consists of the Sinoartial (SA)
node, Atrioventricular (AV) node, the His-Bundle, and Purkinje fibers. The conduction
system is a specialized network of electrical cells in the heart that stimulate the heart to
beat. The electrical impulses from which the heart muscle causes the heart to contract
initiate at the SA node. Once released from the SA node, the impulse causes the atria to
contract. The signal then passes through the AV node, which is located at the junction of
the atria and ventricles. The AV node checks the signal, delaying the activating impulse
and sends it through the muscle Purkinje fibers of the ventricles, causing them to
contract. The AV node serves as a crucial component of the conduction system, as it
coordinates the pumping of the atria and ventricles so that they work together to pump
blood most efficiently and sequentially. 2
Failures in the conduction system, which can ultimately lead to cardiac sudden
death, may result from deficiencies in gene expression within the conduction system.
Because KCR-1 is a neuronal gene, being expressed in the brain and the heart, it was of
particular interest to us. Previous studies in cardiac sudden death have found that genes
found in the brain and the heart, are found in the conduction system within the heart.
Currently, there is little known about the development of the conduction system or what
makes these cells different from the rest of the myocaridum. Recent studies however have
suggested that the cardiac conduction system cells have many of the similar properties as
cells in the brain. KCR-1 was initially identified as a K+ channel regulator in the brain.
Previous studies involving northern blot analysis revealed that KCR-1’s expression is
high in the central nervous system and low in the heart.3 This result was of great
significance because it suggests that the low of expression of KCR-1 may be specific to
the conduction cells. Conduction cells make up only 1% of the population of heart cells,
thus the low expression of KCR-1 in the heart may be specific to these conduction system
cells. We have identified KCR-1 as a conduction system specific gene using in situ
hybridization of adult mouse hearts. Therefore, our hypothesis states that deficiencies in
KCR-1 may result in conduction system defects, such as cardiac sudden death. To test
this hypothesis we first analyzed the general expression pattern of KCR-1. Based on
KCR-1's expression we then designed the construct for the conditional knockouts of
KCR-1. Because we are generating a conditional KO, KCR-1 expression would be
restricted to only certain areas of the conduction system. This will provide the data
needed to see if requirement for KCR-1 comes from cells found in the brain and heart.
Materials and Methods
Whole mount insitus were performed on mouse embryos and isolated mouse
hearts as described by (St. Amand et al., 1998).4 Construction of the KCR-1 vector to
knockout exon 1 was done by PCR amplification and cloning into the pFlox-FRT Neo
(courtesy from Chen) vector. PCR amplification was performed on the exon 1 and cloned
in between 2 loxP sites. Adjacent 4kb 5` sequence was cloned using Xho1, and 4kb 3`
sequence was cloned using Not1/Sac1. Exon 1’s location in between 2 loxP sites will
enable it to be taken out when crossed with Cre.
Results
In situ hybridization was performed to observe KCR-1's expression pattern. In
situ hybridization is the use of an RNA probe to detect a specific RNA sequence within
certain tissues and/or organs.5 Prior studies indicate KCR-1 is expressed in the
conduction system of adult mouse hearts. Therefore, we performed whole mount in situ
to analyze KCR-1’s expression during development. Whole mount in situ hybridization
of the E8.5 –E11.5 mice embryos and E14.5 isolated hearts revealed that KCR-1 is
expressed as early at E8.5 but, expression seemed to be non-specific to a certain area.
This non-specific expression pattern continued to be seen through E11.5.
We were able to get the 5` sequence cloned into the vector, pFlox-FRT-Neo,
however, time constraints prevented us from cloning in the 3` sequence.
Discussion
As a K+ channel regulator being expressed in the heart and the brain, KCR-1 is
thought to play a pivotal role in normal electrophysiological functioning of the heart.
Deficiencies in KCR-1 may result in conduction system defects, such as cardiac sudden
death. Due to KCR1’s non-specific expression pattern as seen in the mouse embryos, we
see that its widespread expression through development makes KCR-1 an undesirable
marker for looking at development of the conduction system. Due to KCR-1’s expression
in the conduction system as seen in the adult mouse heart, we sought to determine its
function in terms of affecting the onset of cardiac sudden death. This required the
construction of conditional KCR-1 knockout (KO) mice. We suspect that KO of KCR-1
will lead to conduction system defects, such as sudden death. Because we are generating
a conditional KO, KCR-1 expression would be restricted to only certain areas of the
conduction system. This will provide the data needed to see if requirement for KCR-1
comes from cells found in the brain and heart.
“Sudden Cardiac Death”. http://www.americanheart.org/presenter.jhtml?identifier=4741
Aug. 09, 2002
2
“The Conduction System”. Texas Heart Institute. http://www.tmc.edu/thi/conduct.html March
2002
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3
St. Amand, Tara et al.,. “Antagonistic Signals between BMP4 and FGF8 Define the Expression of
Pitx1and Pitx2 in Mouse Tooth-Forming Anlage”. Developmental Biology 2002
5
“In Situ Hybridization”. Immunohistochemistry-In situ Hybridization. http://home.no.net/immuno/.
Jan. 2002
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