You have cloned a new gene X. Patch clamp recording from
HEK293 cells overexpressing X revealed that X-transfected cells express large
ionic currents when these cells are mechanically stimulated (i.e. by touching
the cell with glass rod); no such currents were seen in untransfected cells.
Immunostaining of rat tissues with antibodies against X revealed that X is
highly expressed in a subset of large dorsal root ganglia and trigeminal
ganglia neurons and also in the auditory hear cells. Design the experimental
strategy to test if gene X is an ion channel, what other tissues it is expressed in and its physiological role.
Cells that express ionic currents when one recombinant gene
is implanted but no current in native cells means that this gene is likely an
ion channel. The current direction gives an indication of the role of the gene
as a large downward inflection would suggest a role in depolarisation. However,
without further experimental evidence, a whole cell patch clamp recording
cannot confirm that gene X is an ion channel. The following will discuss the
techniques for identifying whether it is an ion channel, tissue expression and
the function of the X protein.
Is it an ion channel
and what does it conduct?
Before adding the recombinant channel, a green fluorescence
protein tag sequence can be added to the gene. This would allow viewing of the
plasma membrane through Total Internal Reflection Fluorescence Microscopy (TIRF)
which emits light at an angle where the wavelengths are all reflected away from
the specimen but some photons diffuse through and excite fluorophores only on
the PM. So there will only be a strong fluorescent signal from the protein if
it is present on the PM. If this was not the case it is unlikely to be an ion
channel. Using native HEK cells as a control will make for easy comparison.
Ion channels have a distinct single-channel recording
profile so a cell-attached patch clamp to isolate one channel could be done and
record from the single channel whilst mechanically stimulating the cell. An ion
channel would provide a current trace like in figure 1.
Also this technique discounts the chance that
the gene is an ion-pump because a single pump recording is too low to be
detected. siRNA should be used to reduce the expression of the mRNA of the gene
and then redo whole-cell voltage clamp recordings and compare the first
recombinant cell recordings as a control. If the current is significantly
reduced it is likely that the gene codes an ion channel. A control using
scrambled siRNA can be used which as it has no effect on X-mRNA so if the
current still decreases then it is not due to X-mRNA knockdown as this
non-specific action is a limitation of siRNA.
Identifying the ions it conducts can begin with adding NMDG,
if the current is ablated then the channel is a cation channel. If not, it is
likely a chloride channel. A cation channel could be non-selective so the
channel can then be treated with tetrodotoxin (Na+), tetraethylammonium (K+) or
ruthenium red (Ca2+) separately, if the current is reduced by any of these then
the channel conducts that ion. If all do then it is non-selective. However, not
all subtypes of each cation channel are inhibited by these blockers, such as
NaV1.7. Therefore, the results can be confirmed by removing each cation from solution
and measuring the current.
Expression in other
tissues:
Immunohistochemistry and electrophoresis can be used in
conjunction to support the results of each test. Being as this is a newly
cloned gene it is unlikely there is an antibody for it. One could be produced
in normal animal-anti-animal technique or an epitope could be genetically
introduced into the gene of transgenic species. Tissue slices are taken and
stained with the specific primary antibody to bind and then a secondary,
fluorescent antibody. The tissue will then fluoresce under microscope if the
tissue expresses the protein. Occluding primary antibody acts as a control to
show the non-specific binding of secondary antibodies.
SDS-PAGE electrophoresis denatures and adds uniform charge
to the proteins of a sample from each tissue. The proteins are separated by
size then radio or fluorescent antibodies are used on the film which will
appear as a blot under x-ray or microscope if the protein is expressed in the
tissue. The control for this will be to normalise the expression level compared
to actin as it is expressed at similar levels in all tissues.
Testing the
physiological role of gene-X:
It is wise to use siRNA knockdown first because the siRNA
can be delivered to the dorsal root ganglion via viral mediated injection and
reduce the expression levels of the gene so behavioural and physiological
changes can be tested. It is especially useful to do first because the same
individual can be used as the control and experimental group which reduces the
variability in response and reduces the number of animals being used at this
stage. If this stage produces positive results then knockout mice can be used
to compare.
Flanking the gene with LoxP and having KO mice only express
Cre under the NF200 promoter so the gene would only be lost in large diameter
DRG neurons. After doing so it is possible to view any behavioural changes and
because it is likely a mechanically activated ion channel, use tests such as
Von Frey filament to measure changes to innocuous touch. Post-testing, DRG
neurons can be dissected and stained to identify any changes to the neuron
physiology.
Evaluation of this
experimental design:
The methods being used are ones common to this type of research
and yield reliable, reproducible results that are easily interpreted without
the need for extensive normalisation calculations. In addition, the
experimental design that is laid out uses at least two methods for collecting
data so each will support the results from the other or present discrepancies
that can be dealt with rather than drawing false conclusions. Also, with fairly
few experiments, the tissue expression, which ion is conducted, the mechanism
by which the channel is activated (mechanical) and the function can be
identified. However, the specific way mechanical stimuli open the channel is
not identified.
Transfection of genes is only 10% so large cultures are
required for success, also GM and transgenics are expensive and have stringent
guidelines for use. TIRF is also an expensive technique. Another issue is that
low levels of expression in tissues are not detected by these methods. If
required RNA-sequencing could be used instead to detect low levels. Cysteine
linked optogenetics would be useful for identifying the channel function but
can only be used if the channel is ligand activated, as this channel is
mechanical, it cannot be used.
In summary, gene-X can be identified as an ion channel
through distinct traces when measuring single channel currents and using siRNA
to knockdown the gene to view the change in current. The ion it conducts can be
identified by removing specific ions from the solution and measuring the change
in current. Electrophoresis will show the tissues expressing gene-X and
knocking out the gene will identify the function of the gene through the
changes that occur when it is not expressed.
