tag:blogger.com,1999:blog-14855385526869983702024-03-14T03:09:36.046-07:00The Horton LabUnknownnoreply@blogger.comBlogger11125tag:blogger.com,1999:blog-1485538552686998370.post-79502637995841449292013-04-10T09:58:00.001-07:002013-04-10T10:02:38.768-07:00Transgenic mice - research update <div style="margin-bottom: .0001pt; margin: 0in; text-indent: .5in;">
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<span style="font-size: 10pt;">As mentioned earlier, we have been working
on identifying gene targets for soluble FGFR3 cleaved sICD fragment using
ChIP-sequencing. We have made considerable progress in optimizing the protocol
for this experiment and have also initiated our first ChIP-sequencing
experiment. We are beginning to prepare for additional
ChIP-sequencing experiments based on the results from our preliminary
investigation.<o:p></o:p></span></div>
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<span style="font-size: 10pt;">Additionally, the Ach-mutant transgenic
mice line is being established. We have a few different transgenic lines in our
facility that are in the process of being genotyped to confirm their genetic
make-up. Once confirmed, we will start using them initially to perform certain immunohistochemistry
and protein studies. <o:p></o:p></span></div>
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</div>
Unknownnoreply@blogger.com1tag:blogger.com,1999:blog-1485538552686998370.post-72900760252454091362013-01-30T21:45:00.000-08:002013-04-10T09:21:38.890-07:00Research UpdateWe continue to work on ChIP-sequencing. Considering this will be the first time we will be performing a genome wide search for FGFR3 target genes, we have to do a lot of troubleshooting and run preliminary experiments to confirm our experimental conditions. We have made considerable progress and will be performing initial ChIP-sequencing experiments.<br />
<br />
On the other hand, we are also making progress in developing transgenic mice that will be used to discover effects of FGFR3 mutations on bone growth. Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1485538552686998370.post-54720387053767125492012-09-27T15:36:00.006-07:002012-09-27T15:39:25.640-07:00Regulating FGFR3 degradation<!--[if !mso]>
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Down regulation of receptor signaling often involves internalization
of the receptor followed by its degradation. <span style="mso-spacerun: yes;"> </span>Fewer active receptors found on the cell
surface mean lesser capacity for signal. Reduced number of receptors on the
surface also affects the duration of a particular signal. Accordingly, internalization
of FGFR3 will reduce its signaling capacity on the surface and provide an important
regulatory mechanism for decreasing FGFR3 signaling. Internalization of
receptor (by a process called <i>endocytosis</i>) can take place through one or more pathways
as shown in the figure below. </div>
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<br /></div>
<div class="separator" style="clear: both; text-align: center;">
<a href="http://2.bp.blogspot.com/-iRasa5OQa7Y/UGTUr1RI-NI/AAAAAAAAABM/7pXXC5MOsLo/s1600/nrc1146-f2.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="382" src="http://2.bp.blogspot.com/-iRasa5OQa7Y/UGTUr1RI-NI/AAAAAAAAABM/7pXXC5MOsLo/s400/nrc1146-f2.jpg" width="400" /></a></div>
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<div class="MsoNormal">
We have previously shown that FGFR3 can be internalized in a
clathrin/dynamin-dependent manner and this is required for receptor cleavage to
occur that generates the soluble intracellular piece (sICD) (Degnin et al, 2011). Others have reported that
FGFR3 may be internalized by both clathrin-dependent and clathrin-independent
mechanisms. The mechanism of FGFR3 degradation has not been clearly delineated.
We have evidence that FGFR3 forms a complex with proteins, called chaperones
that help keep FGFR3 stable. Upon dissociation from the chaperone complex, FGFR3
gets processed and is degraded. The chaperone complexes seem to be important in
maintaining stable FGFR3 and therefore also become important targets that
affect FGFR3 degradation. </div>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1485538552686998370.post-40192365467627897292012-07-16T19:45:00.000-07:002012-07-18T11:09:33.982-07:00Expanding our current hypothesis<br />
Our current working hypothesis is that sICD, the intracellular cleaved
fraction of FGFR3 is involved in regulating at least some of the FGFR3
functions in bone growth and development. We believe it is doing so in part through
interactions within the nucleus and possibly through interactions with DNA. But there are other aspects involved in FGFR3
regulation of bone growth that are essential and will also need to be focused.
Let us take a look at two such ideas - <br />
<div style="margin-left: .5in; mso-list: l0 level1 lfo1; text-indent: -.25in;">
1.<span style="font: 7pt "Times New Roman";">
</span>Subcellular localization and nuclear translocation
– To function in the nucleus, the sICD first needs to be transported to the nucleus. This transport to the nucleus is likely to be a regulated process. There are two ways that the sICD could get into the nucleus. First, it has within its sequence a nuclear
translocation signal that allows it to go to the nucleus, or second, it is aided by another protein that delivers it to the nucleus. We are yet to identify the
mechanism that lets sICD get into the nucleus. </div>
<div style="margin-left: .5in;">
<br /></div>
<div style="margin-left: .5in; mso-list: l0 level1 lfo1; text-indent: -.25in;">
2.<span style="font: 7pt "Times New Roman";">
</span>That brings us to our next question. Does sICD interact
with other proteins? Protein-protein interactions are known to be important for cellular
regulation. Proteins interact with other proteins to form a complex cellular network, and these networks are responsible to carry out cellular functions. Within these networks, protein interactions relay signals that lead to changes in gene expression. Identifying interacting partners can therefor be very useful and can give us some hint about possible protein functions. Identifying sICD interacting proteins will therefore be important in identifying possible genes that may be regulated by sICD. Some of the cellular pathways that involve FGFR3 have been characterized by others and will be useful as a starting point. </div>
<div style="margin-left: 0.5in; text-indent: -0.25in;">
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We
are in the process of addressing some of these ideas. We will take a look at
the techniques and experiments used to answer these questions.Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1485538552686998370.post-17198388305790483782012-06-22T13:00:00.000-07:002012-06-27T19:29:35.135-07:00SELEX to identify gene targets<br />
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<span style="font-family: "Arial","sans-serif";">We have been
looking at ways to identify genes that are regulated by FGFR3, specifically in
the growth plate. To do so, we are working on a modified version of a technique
called as SELEX (<b style="mso-bidi-font-weight: normal;"><u>S</u></b>ystematic <b style="mso-bidi-font-weight: normal;"><u>E</u></b>volution of <b style="mso-bidi-font-weight: normal;"><u>L</u></b>igands by <b style="mso-bidi-font-weight: normal;"><u>Ex</u></b>ponential Enrichment). This method is typically used to
screen for DNA aptamers/sequences that are preferentially bound by a protein/ligand
of interest. Briefly, a library of DNA sequences is generated which contains at
least some DNA sequences that will potentially bind the protein of
interest. The DNA sequences and the
protein of interest are incubated together to let the protein bind to those DNA
that present a preferred sequence for the protein to bind. The protein bound to
the DNA is then isolated through several wash steps, and finally the specific
DNA that was bound to the protein is identified by regular sequencing. </span></div>
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<a href="http://3.bp.blogspot.com/-IHXYYAmQnYk/T-tlxlXbkhI/AAAAAAAAAA4/Ta45xwpLfe8/s1600/pic1.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="229" src="http://3.bp.blogspot.com/-IHXYYAmQnYk/T-tlxlXbkhI/AAAAAAAAAA4/Ta45xwpLfe8/s400/pic1.png" width="400" /></a></div>
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<span style="font-family: "Arial","sans-serif";"><br /></span><span style="font-family: "Arial","sans-serif";"></span></div>
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<span style="font-family: "Arial","sans-serif";">Further
reading:</span></div>
<ol>
<li><span style="font-family: "Arial","sans-serif";"><span style="font: 7pt "Times New Roman";"></span></span><span style="font-size: small;"><span style="font-family: "Arial","sans-serif";">Roulet, Emmanuelle, Stéphane Busso,
Anamaria A Camargo, Andrew J G Simpson, Nicolas Mermod, and Philipp Bucher. “High-throughput
SELEX SAGE Method for Quantitative Modeling of Transcription-factor Binding
Sites.” <i>Nature Biotechnology</i> 20, no. 8 (August 2002): 831–835.</span></span></li>
<li><span style="font-size: small;"><span style="font-family: "Arial","sans-serif";"><span style="-moz-font-feature-settings: normal; -moz-font-language-override: normal; font-family: "Times New Roman"; font-size-adjust: none; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"></span></span><span style="font-family: "Arial","sans-serif";">Jolma, Arttu, Teemu Kivioja, Jarkko Toivonen,
Lu Cheng, Gonghong Wei, Martin Enge, Mikko Taipale, et al. “Multiplexed
Massively Parallel SELEX for Characterization of Human Transcription Factor
Binding Specificities.” <i>Genome Research</i> 20, no. 6 (June 1, 2010):
861–873.</span></span></li>
<li><span style="font-size: small;"><span style="font-family: "Arial","sans-serif";"><span style="-moz-font-feature-settings: normal; -moz-font-language-override: normal; font-family: "Times New Roman"; font-size-adjust: none; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"></span></span><span style="font-family: "Arial","sans-serif";">Stower, Hannah. “Gene Regulation:
Resolving Transcription Factor Binding.” <i>Nature Reviews Genetics</i> 13, no.
2 (December 29, 2011): 71–71.</span></span><span style="font-family: "Arial","sans-serif";"></span></li>
</ol>
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<span style="font-family: "Arial","sans-serif";"></span></div>
<span style="font-family: "Arial","sans-serif";"></span><span style="font-family: "Arial","sans-serif";"></span>Unknownnoreply@blogger.com7tag:blogger.com,1999:blog-1485538552686998370.post-23279292846979045642012-05-07T10:02:00.000-07:002012-05-07T10:02:03.296-07:00Use of micromass cultures to study how sICD effects cell fate.<!--[if gte mso 9]><xml>
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<span style="font-family: "Cambria","serif"; font-size: 12.0pt; mso-ascii-theme-font: major-latin; mso-hansi-theme-font: major-latin;">Bone, cartilage, and fat muscles are all
formed from a common pool of embryonic mesoderm cells. The type of tissue
(bone, fat or cartilage) that develops depends on specific stimulation that is
produced through cellular signals. Signals involved in bone formation
(osteogenesis), cartilage (chondrogenesis) and fat muscle (adipogenesis) are
not well known.<span style="mso-spacerun: yes;"> </span>Shown in the diagram
below, we plan to use </span><span style="font-family: "Cambria","serif"; font-size: 12.0pt; mso-ascii-theme-font: major-latin; mso-bidi-font-family: "Times New Roman"; mso-hansi-theme-font: major-latin;">murine C3H10T1/2 cells to analyze the effect of sICD on </span><span style="font-family: "Cambria","serif"; font-size: 12.0pt; mso-ascii-theme-font: major-latin; mso-hansi-theme-font: major-latin;">osteogenesis, chondrogenesis, and
adipogenesis. We will further look at the genes that are affected by these
assays to identify potential regulators of these cellular differentiation
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<br />Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1485538552686998370.post-37263649223499429612012-03-27T11:40:00.001-07:002012-03-27T11:40:19.727-07:00Transgenic mice for FGFR3 signaling in vivo – Part II<!--[if gte mso 9]><xml>
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<br />
<div class="MsoNormal">
Research Update: </div>
<div class="MsoNormal">
</div>
<div class="MsoNormal">
As mentioned in the previous blog, we are in the process of generating
different transgenic mice that will be used to better define the functional
role of cleaved soluble intracellular fragment (sICD) of FGFR3. Specifically,
we are generating three types of transgenic mice: the wild-type FGFR3, the
cleavage resistant FGFR3, and the achondroplasia-FGFR3. The wild type FGFR3 is the
normal FGFR3 capable of performing all the normal functions. The
cleavage-resistant FGFR3 has a mutation in its molecular structure that
disables cleavage, and therefore will not be able to generate the sICD fragment
unlike the wild-type FGFR3. The achondroplasia-FGFR3 mice contain the FGFR3
with the same mutation found in Achondroplasia condition. Additionally, we will
also have mice that express only the sICD fragment, and will be used for future
experiments. The process of generating and establishing these transgenic mice involves
several steps as discussed in the previous blog. We are currently validating
and preparing assays to genotype (the process of determining/identifying the
specific genetic type of transgenic mice) mice that will be generated. Once
established, these transgenic mice will be used in different experiments that
will reveal functional roles for sICD in growth plate development. </div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1485538552686998370.post-33388356091554655252012-02-20T17:26:00.001-08:002012-02-21T07:36:31.092-08:00Transgenic mice to study FGFR3 signaling in vivo – Part 1<span class="Apple-style-span" style="font-family: Times, 'Times New Roman', serif;"><span class="Apple-style-span" style="line-height: 16px;">
</span></span><br />
<span class="Apple-style-span" style="font-family: Times, 'Times New Roman', serif;"></span><br />
<div class="MsoNormal">
<span style="font-family: Georgia, 'Times New Roman', serif;">FGFR3
is part of a complicated network that is required for normal bone growth and
development. Achondroplasia-associated mutation increases the negative role of
FGFR3 (anti-proliferative), causing growth inhibition. Although FGFR3 appears
to have an anti-proliferative role in bones, it has been shown to promote
proliferation in other tissues. So, what makes FGFR3 function differently in
different tissues, and why is it important for us to understand this? To be
able to identify and test molecular targets for drug, we have to find
out how does Ach-associated FGFR3 mutation cause increased growth inhibition in
bones. We need the sequence of events downstream of FGFR3 signaling that are
negatively affected by this mutation. By comparing proliferative vs inhibitory
role of FGFR3, we might get some indication of what is happening in bones due
to the mutations. All of this requires dissecting out the molecular details of
FGFR3 signaling and regulation. Although the initial experiments need to be
carried out in cultured cells, they will need to be eventually tested
in live animals - genetically engineered mice in our case. Our plan is to
use such mice (transgenic mice) models to understand the role of
FGFR3 signaling in bone growth. First, it will be useful to understand what are
transgenic mice and how are they generated.</span><br />
<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;"><br /></span><br />
<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;">Transgenic
mice models are frequently used in the study of human diseases due to their
genetic similarities with humans. They are easy to manipulate and work with.
Most importantly, they provide a method to study the effect of a single gene or
protein without disturbing any other factors in the animal. In simple terms,
transgenic mice expressing the mutant form of a disease-related gene would
mimic the conditions associated with the human disease, and thus allow us to
observe and study the effect of that mutation in the system.</span><br />
<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;"><br /></span><br />
<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;">Generation
of transgenic mice involves several steps. Foreign genetic material - the
transgene - is incorporated into the genome of a mouse to generate transgenic
mice. There are several methods available for the method of delivery of the
transgene and efficient incorporation of the transgene into the genome.
Following link (2 part video) provides an overview of how transgenic system
works. <a href="https://mail.apptix.net/owa/redir.aspx?C=8e2064c7f45a4535ade9c38e316fc101&URL=http%3a%2f%2fwww.youtube.com%2fwatch%3fv%3dujZHrR1mro8"><span style="color: blue; text-decoration: none;">http://www.youtube.com/watch?v=ujZHrR1mro8</span></a></span></div>
<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;"><br /></span><br />
<div class="MsoNormal">
<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;">Also,
for further reading:</span></div>
<div class="MsoNormal">
<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;">1. <a href="https://mail.apptix.net/owa/redir.aspx?C=8e2064c7f45a4535ade9c38e316fc101&URL=http%3a%2f%2fwww.ncbi.nlm.nih.gov%2fpubmed%3fterm%3d%22Van%20Keuren%20ML%22%5BAuthor%5D"><span style="color: windowtext; text-decoration: none;">Van Keuren ML</span></a> et
al. “Generating transgenic mice from bacterial artificial chromosomes:
transgenesis efficiency, integration and expression outcomes.” Transgenic Res.
2009 (<a href="https://mail.apptix.net/owa/redir.aspx?C=8e2064c7f45a4535ade9c38e316fc101&URL=http%3a%2f%2fwww.ncbi.nlm.nih.gov%2fpmc%2farticles%2fPMC3016422%2f%3ftool%3dpubmed"><span style="color: windowtext; text-decoration: none;">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3016422/?tool=pubmed</span></a>)<o:p></o:p></span></div>
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<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;">2.
Connelly CS et al. “The role of transgenic animals in the analysis of various
biological aspects of normal and pathologic states.” <span style="color: #111111;">Exp Cell Res.</span> 1989. (<a href="https://mail.apptix.net/owa/redir.aspx?C=8e2064c7f45a4535ade9c38e316fc101&URL=http%3a%2f%2fwww.ncbi.nlm.nih.gov%2fpubmed%2f2670592"><span style="color: windowtext; text-decoration: none;">http://www.ncbi.nlm.nih.gov/pubmed/2670592</span></a>)<o:p></o:p></span></div>
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<br /></div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1485538552686998370.post-45805032566617100212012-02-20T17:25:00.001-08:002012-02-21T07:36:43.893-08:00More on ChIP to identify DNA-protein interactions<span class="Apple-style-span" style="color: #990000; font-family: Times, 'Times New Roman', serif;">
</span><br />
<span class="Apple-style-span" style="color: #990000; font-family: Times, 'Times New Roman', serif;"></span><br />
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<span class="Apple-style-span" style="color: #990000; font-family: Georgia, 'Times New Roman', serif;"><span class="Apple-style-span" style="color: black;">Progress:</span><span class="Apple-style-span" style="color: black;"><o:p></o:p></span></span></div>
<span class="Apple-style-span" style="color: #990000;"><span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;">
</span></span><br />
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<span class="Apple-style-span" style="color: #990000;"><span class="Apple-style-span" style="color: black; font-family: Georgia, 'Times New Roman', serif;">Our current focus is to identify a role for sICD
(the small intracellular protein fragment formed by the cleavage of FGFR3
receptor) using chromatin immunoprecipitation (ChIP). We would like to find out
whether sICD protein could interact with DNA to change the genetic environment
of the cell and alter the outcome of FGFR3 signaling. We have conducted
preliminary experiments to establish the ChIP method to look at DNA-protein
interactions in cartilage cells. These ChIP experiments will be combined with
ChIP-sequencing analysis to recognize a functional role for sICD protein.</span></span></div>
<span class="Apple-style-span" style="color: #990000;">
</span><br />
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<span class="Apple-style-span" style="color: #990000; font-family: Georgia, 'Times New Roman', serif;"><span class="Apple-style-span" style="color: black;"><br /></span></span><br />
<span class="Apple-style-span" style="color: #990000; font-family: Georgia, 'Times New Roman', serif;"><span class="Apple-style-span" style="color: black;">ChIP-sequencing is a powerful tool that gives
information on DNA-protein interaction sites on a genome-wide (in the whole
organism) level. Briefly, ChIP isolates pieces of DNA that are occupied (or
bound) by a transcription factor (typically a protein) within the whole genome.
The isolated pieces of DNA represent various sites within the whole genome that
the protein binds. Loosely speaking, if a protein occupies or binds to a
certain region (or site) of a DNA, it is probable that it may participate in
regulating expression of that gene. The information encoded by these pieces of
DNA is determined by </span></span><span class="Apple-style-span" style="color: #990000; font-family: Georgia, 'Times New Roman', serif;"><i><span class="Apple-style-span" style="color: black;">sequencing </span></i></span><span class="Apple-style-span" style="color: #990000; font-family: Georgia, 'Times New Roman', serif;"><span class="Apple-style-span" style="color: black;">simply by determining the arrangement of
the nucleotide bases adenine (A), thymine (T), guanine (G), and cytosine(C)
within that DNA. ChIP-sequencing will provide valuable information and lead to
better understanding of the system. More on ChIP-sequencing can be found here:</span></span></div>
<span class="Apple-style-span" style="color: #990000;">
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<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;"><a href="http://en.wikipedia.org/wiki/Chip-sequencing"><span style="text-decoration: none;"><span class="Apple-style-span" style="color: black;">http://en.wikipedia.org/wiki/Chip-sequencing</span></span></a><span class="Apple-style-span" style="color: black;">.</span></span></div>
<span class="Apple-style-span" style="color: black; font-family: Georgia, 'Times New Roman', serif;">In our next blog
update, we will present a second research that is being conducted in the lab
using transgenic mice to help characterize the role of FGFR3 signaling in
achondroplasia</span>
</span>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1485538552686998370.post-12409002848736714082012-02-20T17:24:00.001-08:002012-02-21T07:36:49.598-08:00The progress on ChIP<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;"><span class="Apple-style-span" style="color: #660000;">PROGRESS:</span> We have adapted the ChIP assay to
cartilage cells, which will help us to identify "target genes" that
are regulated by nuclear FGFR3 and potentially involved in
achondroplasia. </span><br />
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<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;"><span class="Apple-style-span" style="color: #990000;">TECHNICAL NOTE:</span> The genetic material of a cell is found inside the
nucleus and is stored in the form of DNA, which is organized into linear
segments called genes. Proteins referred to as transcription factors
bind to DNA and turn the genes on and off. We think the sICD portion
of FGFR3 (see earlier blog) acts as a transcription factor for genes involved
in achondroplasia and the ChIP assay will be used as a tool to identify them.
ChIP stands for <u>Ch</u>romatin <u>I</u>mmuno<u>p</u>recipitation.
The ChIP technique allows us to isolate pieces of DNA that are bound
by the protein of interest - the FGFR3-sICD in this case. For our
experiments, we will analyze cultured chondrocyte cells using ChIP to first
show that the sICD fragment binds to DNA. Once the region of the DNA to which
sICD binds to is identified, we will then determine which specific genes bind
to and are presumably regulated by the sICD. </span><o:p></o:p></div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1485538552686998370.post-29189330861274553852012-02-20T17:23:00.000-08:002012-02-21T07:37:19.037-08:00Achondroplasia - an introduction to our approach<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;">As
discussed under <i>Research</i>, the root cause of achondroplasia is <b><i>too
much</i></b> “anti-growth” activity of FGFR3 in the cells of growing
bones. That is, increase in FGFR3 activity within the cells of growing
bones results in specific signals that reduce or block growth. Several
therapeutic strategies have been proposed to reduce this activity, and most are
targeted to the cellular networks that carry instructions from FGFR3 to nucleus
within the cells. These networks are complex and dispersed throughout the cell.
We have discovered a new pathway that bypasses these established networks and
potentially allows instructions to be transferred directly from FGFR3 residing
on the surface of a cell to its nucleus, where the regulation of genes involved
in controlling growth occurs. We believe this novel pathway mediates many
of the anti-growth signals of FGFR3, including those responsible for
achondroplasia. We also believe that defining this new pathway will lead
to the identification of new and potentially better targets for therapeutic
intervention for achondroplasia.</span><br />
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<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;"><br /></span><br />
<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;">Indeed,
we have already identified one potential target. A key feature of this
new pathway involves cutting of the intact FGFR3 molecule present at the cell
surface to produce a small fragment that moves to the nucleus to deliver its
anti-growth message, and promotes nuclear signaling of FGFR3. To our
surprise, the machinery that cuts FGFR3 also cleaves a protein implicated in
Alzheimer’s disease. <i>Other than sharing this common protein cutting
machinery, the two proteins and also the two diseases are completely unrelated</i>.
In fact, abnormal cutting of this protein in many patients with
Alzheimer’s disease has identified this cutting machinery as a therapeutic
target for Alzheimer’s disease, which in turn has led to considerable
investment by the pharmaceutical industry in drugs to correct the abnormality.<o:p></o:p></span></div>
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<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;"><b><br /></b></span><br />
<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;"><b>So
how is this relevant to achondroplasia?</b> As we investigated the cleavage of FGFR3,
we discovered that achondroplastic FGFR3 is cut abnormally creating a scenario
similar to that for Alzheimer’s disease in which abnormal cleavage contributes
to disease. If so, the distinct possibility arises that drugs designed to
favorably alter the cleavage of the Alzheimer protein might also normalize the
cleavage of FGFR3 and at least partially alleviate the manifestations of
achondroplasia. In economic terms, our findings raise the possibility of
leveraging the enormous investment in Alzheimer’s drugs for a novel treatment
for achondroplasia. </span><br />
<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;"><br /></span></div>
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<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;">Our
goal is to move our project from its current stage of initial discovery to the
point where it merits further development by industry. For this we need
to define the new pathway in more depth. We need to demonstrate that this
pathway is involved and necessary for normal FGFR3 effects on growing bone,
that it is altered in achondroplasia, and that restoration of normal cleavage
corrects the disturbance caused by the achondroplasia mutation. In short,
we need to show that the newly discovered pathway influences bone growth, plays
a role in achondroplasia and is amenable to therapeutic manipulation. We
anticipate reaching these goals over the next 2-3 years.</span></div>
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<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;"><br /></span><br />
<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;">We
will discuss how we are addressing these points in future blogs. However,
for this blog suffice it to say that many of our experiments are being
performed in cultured cells. We have developed and continue to refine
assays that measure precisely how much of the cleaved FGFR3 fragment reaches
the cell nucleus. By comparing normal FGFR3 to FGFR3 bearing the
achondroplasia and other relevant mutations, we can begin to understand the
impact of the mutations on the fate of the cleaved fragments. We are also
setting up assays to determine the precise location of cleavage within the
FGFR3 protein and how this is affected by the achondroplasia mutation.
Once this information is known, we will determine if Alzheimer drugs can
normalize cleavage and transfer of the fragment to the nucleus. <o:p></o:p></span></div>
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<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;"><br /></span><br />
<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;">We
expect there to be direct target genes of FGFR3 in the nucleus. To
identify them, we are using a technique called Chromatin Immunoprecipitation
(ChIP). It will be explained in a future blog. Identifying even a
few FGFR3 target genes will provide valuable insights into how FGFR3 regulates
bone growth. Measuring the expression of these genes will further
validate the effects of the achondroplasia mutation and of candidate drugs on
FGFR3 functions. </span></div>
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<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;"><br /></span><br />
<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;">To
complement our cell culture studies, we are also genetically engineering mice
to validate the importance of the FGFR3 nuclear signaling pathway in live
animals. FGFR3 in these mice will be tagged to allow microscopic and
biochemical detection. The mice will have normal FGFR3, FGFR3 bearing the
achondroplasia mutation, FGFR3 bearing a mutation that blocks cleavage and
prevents the fragment from being produced and FGFR3 comprised of only the
cleavage fragment destined for the nucleus. By analyzing and comparing
these mice in detail, we will determine the biologic significance of the direct
nuclear signaling by FGFR3. These mice will also be used to test the
Alzheimer’s drugs.<o:p></o:p></span></div>
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<span class="Apple-style-span" style="font-family: Georgia, 'Times New Roman', serif;">As
noted above, future blogs will focus on progress within the different aspects
of this overall investigation. <o:p></o:p></span></div>
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<br /></div>Unknownnoreply@blogger.com0