Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
Vertebrate Genetics, Fall 2012
Nirao Shah
nms@ucsf.edu; 4-4381; Rock hall 348B
Outline of today’s lecture
Intro to olfaction, contd
Gene targeting in the mouse with ES cells: knock-outs, knock-ins
Visualizing an olfactory map in the brain
Maintenance of the olfactory map
Formation of the olfactory map
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
OLFACTION
How does OR identification aid the problem of understanding how we smell particular odors?
ISH of individual OR encoding genes revealed a particularly elegant solution to the problem of
odor recognition. Each sensory neuron in the nose expresses only 1 OR, and each OR is
expressed by only ~0.1% of the neurons in the nose (in a seemingly stochastic pattern). This
expression was subsequently confirmed by single cell RT-PCR - individual olfactory neurons
only express 1 receptor encoding gene. Why is it necessary to confirm the ISH result with RTPCR? Because ISH is not as sensitive as RT-PCR.
So “the problem of distinguishing which OR has been activated by an odor is reduced to the
problem of distinguishing which sensory neuron has been activated.” Why is the problem
simpler now? Because we can ask: where do the olfactory sensory neurons expressing a given
OR project in the brain. In other words, we can begin to map how the brain represents odor.
I7
J7
OMP
In situ hybridization to detect mRNA expression of ORs I7 and J7 and OMP, a gene expressed in
all neurons in the nose. (A, D, G), (B, E, H) and (C, F, I) are cryosections through the nose
labeled with radioactive anti-sense probes for I7, J7 and OMP, respectively. Both sides of the
nose are shown and for orientation, the top of each panel is the top of the nose.
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
ISH to detect message for I7. Cryosection through the neurons in the nose labeled with a nonradioactive anti-sense probe for I7. Three I7+ neurons are labeled in this section.
How do you find out where olfactory sensory neurons expressing a given olfactory receptor
project to in the brain?
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
GENE TARGETING WITH MOUSE EMBRYONIC STEM CELLS (ES CELLS)
Overview
These are pluripotential cells, ie in the appropriate context in vivo or in tissue culture they can
give rise to any of the lineages in the mouse, including gametes. (Note: the placenta is about the
only tissue to which ES cells do not contribute.) They are easily propagated as undifferentiated
cells in tissue culture, and undergo homologous recombination readily with introduced DNA
sequences that bear regions of homology with endogenous sequences. As such they are
invaluable for gene targeting, the manipulation of a locus by homologous recombination. This
permits deletion of endogenous sequences (knock out) as well as the insertion (knock in) of
heterologous DNA (see below).
ES cells are derived from the inner cell mass (ICM) of the blastocyst, which gives rise to all the
tissues in the embryo. The trophoectoderm is considered to constitute extra-embryonic lineages,
and it forms supporting structures (placenta...). See figure below....
not to scale.
ES cells were derived by Evans (’81) and by Gail Martin (’81) at UCSF. ES cell lines are
maintained as low copy passages and are periodically checked for euploidy (2n). Most ES cell
lines are derived from 129/SvEv labmice, which have a brown coat color. Commonly used ES
cells are derived from male blastocysts since ES cells from female typically lose an X chr to
become XO.
Two properties of ES cells permit their use in gene manipulation  ability to undergo homologous recombination (see above)
 ability to “take over” blastocyst upon reinjection into the blastocoel (blastocyst cavity).
In other words, ES cells are very good at competing with the endogenous ICM of a
blastocyst, thereby contributing to all the tissues in the resulting chimeric progeny
(except the placenta as noted above). Good ES cell lines are able to contribute to the
germline in a substantial manner, permitting the genome of the ES cells to be transmitted
to the next generation. This = germline transmission of the ES cells you’re using. What
is the frequency of transmission of your targeted allele if your ES cells have gone
germline? What if the targeted locus is X linked and the progeny of the chimera are
female?
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
129 cells (shorthand for 129/SvEv) endow golden brown hair pigmentation which is autonomous
when injected into B6 (shorthand for C57Bl/6J) blastocysts. B6 pups are black. So if you inject
your 129 cells into a B6 blastocyst, what does it mean if you get  pups with all black coats
 pups with variegated black + brown coats
 female pups with brown coat
 all brown male pups?
Generating DNA constructs for gene targeting
Obtain genomic DNA that flanks the region to be manipulated. The genomic DNA may be
obtained by 


ordering a BAC clone spanning the region of interest;
by screening a genomic library containing the region of interest;
PCR of the appropriate genomic region.
It is critical to obtain this genomic DNA from tissues of the same mouse strain as that from
which the ES cells you will use have been derived. Why is this important? Because mouse
strains differ from each other in genome seq, and even a few SNPs can lead to a drop in the
frequency of homologous recombination. If you obtain the region by PCR what would be an
important cross-check? Sequence your product to make sure you have not introduced mutations
in the DNA that you will use for homologous recombination.
Unlike homologous recombination in yeast you need significant homology flanking the mutation
to be introduced into the genome by homologous recombination in ES cells.
From Hasty, Mol Cell Biol 11:5586, 1991
Gene targeting frequencies vary between loci for poorly understood reasons. Nevertheless a
homology of ≥ 4 kb on 1 arm and ≥ 1.5 kb on the other arm appears to work for most genetic
loci. “Arms” are the homologous DNA sequences flanking the mutation you are generating.
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
As an example, in the figure shown below, we wish to generate an in-frame fusion of EGFP at
the C-terminal of a protein of interest (only the last 2 exons, # 11 & 12, are shown). This should,
in theory, permit tagging of the functional protein with EGFP.
We use an ubiquitous promoter (UP) to drive expression of neomycin resistance, and the neo has
its own stop codon (not shown) and pA signal. The presence of neo in the targeting vector
permits the selection of ES cells that have incorporated your construct. In this case, we have
used ~2.2 kb homology for the 5’ arm and ~5 kb homology for the 3’ arm, which should provide
ample homology for recombination in ES cells.
Note that the neo cassette is flirted (flanked by FRT sites). This will permit the eventual deletion
of the neo selection cassette. Why is this important? The neo is driven by its own promoter and
utilizes a pA different from the endogenous locus. The potential of disrupting transcription &/or
stability of the endogenous gene (now fused to EGFP, of course) is very high if we leave the neo
in place. This becomes problematic if a lof allele is not desired.
not to scale
How do you genotype the resulting neo resistant ES colonies? Typically PCR, using primers that
can only amplify homologously recombined DNA. See figure below. The targeting frequency
(ie freq of homologous recombination events) varies from <1% to >50%, depending primarily on
the locus and the line of ES cells. Why never close to 100% targeting frequency? Because
random integration much more common, and selecting for neo resistance does not distinguish
between random integration and homologous recombination.
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
If you now wish to target the locus again, must use appropriate homology arms and a different
selection regime. Hygromycin or puromycin are viable alternates to neo. Rarely can get both
alleles targeted even with just one round of electroporation.
To obtain mice bearing the homologously recombined modified allele  Thaw targeted colony & expand. (Freeze backup stocks!)
 Inject ES cells into the ICM of recipient blastocysts (129 cells into B6 blastocysts).
 Transfer injected embryos into recipient females
 Pups born ~15 days later. Why only 15 days? Because blastocyst itself already ~3 - 4
day post fertilization.
 Coat color of pups (visible ~ 7 days after birth) indicates degree of chimerism; in our case
all brown male pups would make us very happy.
Mate chimera to wildtype females. Once your chimera has transmitted the modified allele
through the germline, you would typically cross the heterozygous mouse with a mouse strain
expressing Flpe (Flp enhanced for functionality in mammals, ie at 37C) in all tissues. This
would lead to excision of the neo cassette, and the resulting progeny would now bear the flped
(aka flped out) allele homologously recombined into the locus of interest. You would of course
re-genotype to ensure correct excision of the neo.
For modified genomic locus: Solid gray arrows represent primers to PCR genotype 3’ arm; empty block arrows
represent primers to PCR genotype 5’ arm. The PCR products would be unique in each case to homologous
recombination events only.
For modified & Flped genomic locus: Empty block arrows as above. Solid black and gray arrows represent primers
to PCR genotype the neo-deleted 3’ arm.
Dotted line on either end of the locus represents the endogenous genomic sequence into which the targeting vector
integrated by homologous recombination.
Will all good chimeras transmit the targeted allele? No. Degree of chimerism is determined by
checking coat color, and this may not always correlate with the germline contribution of the ES
cells.
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
MAPPING OLFACTORY RECEPTOR SENSORY NEURON PROJECTIONS IN THE
MOUSE BRAIN
How would you go about mapping projections of olfactory sensory neurons?
Could generate a transgene driving expression of a reporter such as EGFP or lacZ....but when
these expts were first performed the promoters that regulated the expression of OR encoding
genes were largely undefined.
Could generate an EGFP fusion to an OR coding gene by gene targeting so that all neurons
expressing the tagged OR could now be visualized. Similar strategy was tried, and it
dramatically affected OR stability, thereby precluding an analysis of the projections of the
sensory neurons.
To map projections of a given OR expressing neurons, Mombaerts et al used a neat trick (see
figure below). They inserted a reporter gene, tau:lacZ, encoding the fusion protein tau:ßgal
immediately 3’ of the stop codon for the gene encoding P2 (an OR). More about the tau:lacZ
later. To ensure expression of tau:ßgal in neurons that express P2, they also inserted an IRES
sequence interposed between the stop codon and tau:lacZ. IRES (Internal Ribosome Entry Site)
permits cap-independent translation of any mRNA found 3’ of the IRES sequence. IRES needs
to be transcribed in order to initiate translation of the downstream mRNA. This strategy
therefore permits translation of the tau:ßgal fusion in all (and only in these) neurons expressing
P2.
Tau normally associates with cytoskeletal proteins such as tubulin and microtubules, and in
neurons, it is preferentially transported to axons (and not dendrites). Since olfactory sensory
neurons project over quite a large distance (up to 1 cm) to their target sites in the brain, using a
tau:ßgal fusion protein should label the target sites robustly. In fact, this is exactly what was
observed - the axons of sensory neurons expressing P2 are easily visualized and appear to
converge to discrete spots in the brain (more detail than you want......to discrete spots in the brain
called glomeruli located in the main olfactory bulb).
This allele can also be referred to as a P2-IRES-taulacZ allele.
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
This allele, referred to as P2-IRES-tau:lacZ, is a “knock-in” allele, since you have inserted
(knocked-in) a cassette into the P2 locus. Each OR gene that was targeted in a similar manner
revealed discrete & unique convergence points in the main olfactory bulb. So there appears to be
a spatial map in the brain of OR projections....neurons expressing different ORs converge to
distinct locations in the brain. So the problem of figuring out which sensory neurons are
activated by particular odors is now reduced to figuring out which particular spots in the brain
are activated by such odors.
Note that tau:EGFP fusion should also do the same thing, except now the axons should be
fluorescent under the right illumination....and this is borne out by targeting tau:egfp to OR genes
as well. Finally, this is a general way to highlight the axons of all neurons; indeed other labs
have used tau:lacZ to label their favorite neurons. Another commonly used compartmentalized
reporter gene is nuclear lacZ (nlacZ), which has a nuclear localization sequence from the SV40
virus pasted in the N-terminal of ßgal. A histone:EGFP fusion is also commonly used to localize
EGFP exclusively to the nucleus. Big advantage of compartmentalizing a reporter is that it
greatly increases the sensitivity of your assay for the cellular compartment that has been tagged.
Does targeting the IRES-tau:lacZ cassette mess up the axons of the sensory neurons? Is
tau:lacZ faithfully transcribed exclusively in the P2 population? These are important control
expts of course because you’ve introduced heterologous DNA sequences into a gene. ISH
experiments confirm that the P2 neurons are not mis-targeting because of tau:ßgal expression,
and that the expression of the fusion protein is restricted to the P2 population.
P2-IRES-tauLacZ/+ mouse: Whole mount preparation of the nasal cavity and brain stained for gal activity (Xgal).
Panels d, e are higher mag views of the olfactory sensory neurons in the nasal cavity (d) and their terminations in
the olfactory bulb in the brain (e). Modified from Mombaerts, 1996.
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
P2-IRES-tauLacZ/+ mouse: Faithful projections of
P2 olfactory sensory neurons to their target in the
glomerular layer. All ORs are mono-allelically
expressed (ie a single sensory neuron not only
expresses a single OR gene, but it also expresses only
one of the two alleles. So both P2 alleles are
expressed, just in different neurons and all P2
neurons project to the same target in the olfactory
bulb.).
Can use the property of mono-allelic
expression to compare projections of neurons
expressing the WT and the modified P2 alleles.
Result: they overlap, demonstrating that the genetic
modification of the P2 locus has not disrupted
expression of P2.
Panel e shows a section through the olfactory bulbs
labeled with radioactive in situ hybridization for P2
mRNA; P2 is transcribed at very high levels,
permitting visualization of the message even in the
projections of P2 neurons to the brain. Panel f is a
directly adjacent section to that in panel e, and it is
stained for gal activity using Xgal. Panels g and h
are higher mag photos from panels e and f, and they
show 1:1 correspondence between the projections
labeled for P2 mRNA and gal.
Top panel is a schematic of a cross-section of the two
olfactory bulbs so that the top of the panel
corresponds to the top, dorsal side of the brain.
Where the two bulbs abut is the midline.
Modified from Mombaerts, 1996.
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
MAINTENANCE OF OLFACTORY PROJECTIONS TO THE BRAIN IN THE FACE
OF CONTINUAL TURNOVER OF THE SENSORY NEURONS
There is continual turnover of olfactory sensory neurons. The subset of dying neurons is
replaced by a reservoir of olfactory stem cells in the nose.
The olfactory projections to the brain are thought to form right around birth. If these sensory
neurons die eventually, how do the newly born sensory neurons project to the correct
convergence point in the brain? Correct convergence is important to maintain the fidelity of the
spatial map of odor coding.
There are two possibilities that could explain accurate convergence in adult life from newborn
olfactory sensory neurons:
 Newly generated neurons could send their axons along the already laid projections of preexisting neurons expressing the same OR.
 The newly generated neurons could utilize the same mechanism used during development
to guide their projections to the brain. In other words, the developmental guidance cues
perdure into adult life.
How to test this?
If genetically ablate all olfactory sensory neurons expressing a given OR then  Should get no convergence if the adult projections need the pre-laid projections of
neurons born earlier.
 Should get convergence if use pre-existing developmental cues.
So need a way to ablate all neurons expressing P2 (for example) in the adult animal.
Use the reverse tetracycline controlled transactivator (rtTA) system, which only activates gene
transcription in the presence of doxycycline (see figure next pg). Use the rtTA responsive tetOPmin sequence to control the expression of a cell autonomous toxin. In this case, use diphtheria
toxin subunit A (DTA), which irreversibly inactivates EF-2 (elongation factor 2) in the cell,
thereby inhibiting translation. DTA is universal cell lethal toxin. Note that doxycycline (dox) is
chemically similar to tetracycline, is better tolerated by animals, and can penetrate tissue barriers
(such as the blood brain barrier) more effectively than tetracycline.
Constructs & knock-in and transgenic mice:
P2-IRES-rtTA-IRES-tau:lacZ; tetO_Pmin-DTA-pA.
The neo cassette for the knock-in allele is implicit (as is its removal before analyzing
phenotypes); don’t need neo or other selection marker for the transgenic allele of course.
Adult supplementation with doxycycline should do the trick.
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
Schematic outline of the Tet regulatory systems. (a) Mechanism of action of the tetracyclinecontrolled transactivator (tTA). The gene for the repressor (tetR) of the E. coli Tn10 tetracyclineresistance operon is fused to a DNA sequence encoding a carboxy-terminal portion of protein 16
of herpes simplex virus (VP16) that functions as a strong transcription activator. A fusion
product, tTA, can be obtained that binds in the absence of Dox, but not in its presence, to an
array of seven operator sequences (tetO) and activates transcription from a minimal human
cytomegalovirus promoter (Pmin), which itself is inactive. The combination tetO–Pmin is
defined as Ptet. Tissue-specificity of the system is achieved by placing the expression of the tTA
gene under the control of a tissue- or cell-specific promoter (Psp), and consequently tTA will
activate transcription of the candidate gene (X) only in restricted cell populations in a Doxdependent manner. (b) The rtTA factor is derived from tTA: the exchange of three amino acids
within the tetR moiety has converted the phenotype of the transactivator, which now requires
Dox for binding to tetO. Pmin is thus active in the presence, or inactive in the absence of Dox,
respectively. Here again, tissue-specificity of the system is achieved by placing the rtTA gene
under the control of a tissue-specific promoter (Psp). In newer versions of the transactivators, the
VP16 moiety has been replaced by minimal activation domains that have eliminated the
disadvantages of VP16.
From Mansuy, et al 2000.
In these experiments by Gogos et al, feeding the mice doxycycline leads to death of P2expressing neurons, as indicated by disappearance of tau:ßgal staining. Subsequent to
doxycycline withdrawal, P2-expressing neurons are born, and these appear to converge to the
appropriate place in the brain in the absence of pre-existing connections of P2 neurons. Thus,
olfactory sensory neurons are likely to utilize pre-existing guidance cues to reach their targets in
the brain.
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
Here’s some of the data from this study:
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
THE ROLE OF ORs IN FORMING PROJECTIONS OF OLFACTORY SENSORY
NEURONS TO THE BRAIN
The data from the DTA-kill expt discussed above suggests that the sensory neurons in the nose
can navigate back to their target site in the adult brain even in the absence of pre-existing
neurons expressing the same OR. What guidance mechanism(s) is the neuron using to send
projections back into the brain? In the simplest model, olfactory sensory neurons utilize the OR
on their plasma membrane to converge on to their target site in the brain. How do you test this
idea? Knock out (delete) a gene encoding an OR  If the neurons use the OR as a guidance receptor then should see neurons losing their way
 If this is not the case then neurons should maintain their connectivity to the brain in the
absence of functional OR on their surface.
Generate the following deletion (knock-out) allele:
P2-IRES-tau:lacZ
(The  is often used to refer to a deletion; note that this is a knock-out, knock-in allele, since
you’ve deleted the P2 coding seq and inserted a reporter cassette).
In mice bearing this allele, tau:ßgal+ axons were observed to enter the brain but these axons
never converged on to their target site, and eventually completely disappeared.
Consistent with role for ORs in guiding neurons to the correct location in the brain.
P2deletion-IRES-taulLacZ
P2-IRES-tauLacZ
Deletion of P2 (an OR) abolishes projections of P2 neurons to the olfactory bulbs. True in homozygotes and
heterozygotes.
What else do you need to show to convince a critical reader?
Is the OR found on the business end of axons as they enter the brain? Important to know this,
since if ORs sense guidance cues for pathfinding, then they must be found on the plasma
membrane of the pathfinding axons, including at the most distal (furthest from cell body) axonal
tips. Antibodies specific to one or the other ORs demonstrate this convincingly.
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
Is this sufficient evidence that ORs act as guidance receptors to help olfactory sensory neurons
track to the olfactory bulb?
The above result is also consistent with the notion that ORs are simply acting as survival factors
for neurons. In other words, a separate guidance system leads the olfactory sensory neurons to
the correct location and ORs subsequently act to allow these correctly targeted neurons to live.
Thus, a gene product (in this case, the ORs) can be necessary for a process but it may not itself
play an active role in the underlying mechanism; in these cases, the gene product is said to play a
permissive role in the process. How would you test the contrasting scenario in which the OR
plays an instructive role in olfactory sensory neuron guidance to the olfactory bulb? You ask
whether the OR is sufficient to guide these neurons to their targets in a “heterologous” setting.
Various OR substitution constructs were designed:
 M12(P2)-IRES-tauLacZ
 M50(P2)-IRES-tauLacZ
 P3(P2)-IRES-tauLacZ, etc
Note that each section is cut at 20µm thickness, so that the novel M50(P2 )-IRES-tauLacZ target is ~250 µm away
from the endogenous P2 target. While the endogenous M50 target is very close to the novel M50(P2)-IREStauLacZ target in this axis, it is located much closer to the brain surface compared to the novel target, which is
~300 µm deeper in the brain. Arrows in F point to the novel target and arrowheads in F point to the endogenous
target of M50 expressing neurons. “glomeruli” is the anatomical term for the target of olfactory sensory neurons.
In each case, the olfactory sensory neurons expressing M12 (or M50 or P3) instead of the
endogenous P2 tracked to a distinct location in the olfactory bulbs: distinct from the WT P2 or
the WT M12 locations. Suggests that ORs play an instructive role in the process, but that they
are not the sole guidance mechanism.
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Nirao Shah
Genetics 200A: Vertebrate genetics, lecture 2
ADDITIONAL READING
Olfactory related
A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.
Buck, et al. Cell. 1991 Apr 5;65(1):175-87.
A zonal organization of odorant receptor gene expression in the olfactory epithelium. Ressler et
al. Cell 1993 73:597-609.
Spatial segregation of odorant receptor expression in the mammaliam olfactory epithelium.
Vassar et al. Cell 1993 74:309-318.
Visualizing an olfactory sensory map.
Mombaerts, et al. Cell. 1996 Nov 15;87(4):675-86.
Odorant receptors govern the formation of a precise topographic map.
Wang, et al. Cell. 1998 Apr 3;93(1):47-60.
Genetic ablation and restoration of the olfactory topographic map.
Gogos, et al. Cell. 2000 Nov 10;103(4):609-20.
Gene switching and the stability of odorant receptor gene choice.
Shykind, et al. Cell. 2004 Jun 11;117(6):801-15.
ES cells
Gene targeting in ES cells. Soriano, P. Annu Rev Neurosci. 1995;18:1-18. Review.
Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first
century. Capecchi, M. Nat Rev Genet. 2005 Jun;6(6):507-12. Review.
Altering the genome by homologous recombination.
Capecchi, M. Science. 1989 Jun 16;244(4910):1288-92. Review.
Establishment in culture of pluripotential cells from mouse embryos.
Evans, et al. Nature. 1981 Jul 9;292(5819):154-6.
Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by
teratocarcinoma stem cells.
Martin, GR. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7634-8
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