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hello my name is dsy I'm a director at
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The maxp Institute for heart and lung
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research in vadheim Germany and today in
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this third part we're going to be
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talking about the phenomenon of genetic
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compensation in the context of vascular
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development in the zeu fish embryo
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historically Gene function in zebaish
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has been studied initially through a
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forward genetic approach mainly for
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mogiz the genome introducing mutations
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randomly and then doing phenotypic
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screening more recently Molinos
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anti-os have been used to nug down Gene
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function and then more recently just a
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few years ago using zinc finger
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nucleuses as well as Talons and Cris 9
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technology mutations have been
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introduced in specific genes to study
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not only genes that were studied
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previously using Molinos but also other
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genes and the studies that I'll be
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telling you about today were essentially
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inspired by the fact that looking at
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specific genes that have been studied
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both using Molinos as well as reverse
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genetic techniques it became apparent
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that in many cases the phenotypes in
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induced by mutations were much milder
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than those induced by the mopina
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technology and so essentially just to
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give you a little background MOS are as
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you can see here modified oligos they're
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highly stable they're B RNA they're used
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to block either translation or
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splicing and so the question then is why
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muttin phenotypes are in fact often
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milder than anti-sense phenotypes so mut
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phenotypes versus anti-sense phenotypes
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I will also be using the word morphant
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for morpholino induced so we'll be
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talking about mutant versus Morphin
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phenotype and mutants are often referred
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to as Knockouts and anti sense as
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knockdowns so let's start by some
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history of anti-sense approach
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anti-sense technology and more than 30
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years ago people working in
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developmental biology were using or
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started using anti-sense RNA
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to essentially block Gene function this
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is both in the context the Frog embryo
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as well as the fly embryo but this
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period was fairly shortlived probably
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because people were concerned about of
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Target effects and so people moved to
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now overexpressing either wild type or
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dominant negative versions of genes or
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proteins and essentially for example in
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this case dominant negative activan
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receptor certainly was known and uh to
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interfere with other proteins besides
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the actimine receptor and yet approaches
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such as this gave us important insights
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into developmental processes and so it's
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important to realize that uh while no
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reagent is perfect suddenly uh these
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reagents can be used to make important
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insights into biological
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processes and so in Ze fish this anti
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Sense Technology using the Minos was
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introduced in 2000 at the same time as
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it was introduced in the frog and a few
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years later a number of guidelines were
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written up essentially to try and as
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best as people could essentially control
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for these Morino studies and
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specifically trying to avoid and
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recognize of Target
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effects and so essentially with this in
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mind and as I said as the uh various
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reverse genetic techniques became
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available people started seeing
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essentially important differences
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between the mutation induced phenotypes
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and the morpholino or anti-sense induced
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phenotypes and this was further
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emphasized by a larger study from Nathan
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Lawson's lab where they essentially
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looked at a large number of genes and
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again found a poor correlation between
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morol induced and mutant phenotypes in
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zebra fish and so essentially this is
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not specific I should mention to the
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zebrafish field in fact if you now look
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at anti- sense work in the mouse is now
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using trans Genesis to drive
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anti-sense transcripts essentially again
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you see a more severe phenotypes from
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the anti-sense approach than from the
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mutation approach and so it's clearly a
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question that spans Beyond or goes
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beyond just using Molinos and probably
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applies to all all anti-sense work so we
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decided to revisit this issue in detail
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and we picked this Gene called egfl7 for
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a number of reasons but mostly because
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in three different settings using the
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anti-sense approach this Gene had been
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implicated in a playing an important
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role in vascular development this was in
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zebrafish in Frog as well as in human
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and etherial cells and yet in the mouse
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mutant there was no pH type not
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discernable
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phenotype just to give you a little
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background about this Gene it encodes an
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ECM protein it's expressed mly by
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endothal cells and it's apparently
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expressed by tumor cells in human
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Cancers and for this reason was the
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direct Target in gench in fact had a
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clinical trial for one of the humanized
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monocon antibody against this
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protein so as I mentioned in zeber fish
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where the first work was done on this
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Gene when you knock down on egfr 7 using
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Morino you see severe defects in number
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of processes including vascular tube
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formation and you also get pericardial
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Eda indicative of failing heart similar
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phenotype was seen in the Frog embryo
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again using anti-sense approach and Al
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well as in human yes cells human and
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atherial cells and as I mentioned the in
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the contrast
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to these uh studies to these findings
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the mouse mutant was in fact
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phenotypically
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normal now this was a little um
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complicated initially by the fact that
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there's a in the hfl 7 Locus as you can
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see here there's a micr micr 126
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embedded in this Locus and this micr is
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also expressed in endothelial cells and
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so in fact the original mutant that was
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made deleted this micro RNA as well and
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this led to the appearance of vascular
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phenotypes but in fact when a specific
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knockout specific mutations were made
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either in the microrna OR egfl7 Gene
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itself it was realized that in fact the
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microrna was the one responsible for the
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phenotype seen in the original mutant so
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essentially the bottom line is that we
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have a severe phenotypes using antisense
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technology in the fish frog and the
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human cells but no phenotype in the
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mouse and so we went on to make a using
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now tailon technology in egfl7
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mutants and we identified a number of
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mutant and focused on two Le one is the
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Delta 3 which removes the proline
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another one is the Delta four that leads
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to a a premature stop codon and this is
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the main alal mutant alal that we'll be
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using for the rest of the study and we
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used high resolution melt analysis to
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develop a a very rigorous and
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reproducible genotyping protocol which
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as your see is essential for the studies
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I'll be telling you
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about so the much like the mouse mutant
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in fact the zish mutant shows a very
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mild if any phenotypes only about 5% of
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the mutant show this transient
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Hemorrhage that you can see here in the
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head of mutant but essentially looking
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at the both the trunk as well as the
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head vasculature essentially one sees no
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discernable or no major phenotype we
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also made an analyze maternal zygotic
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mutants and uh again these were mutants
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this maton zygotic mutants did not show
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a more severe phenotype than just the
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zygotic
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mutants so essentially as I said we now
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have a situation where the mutants in
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zebra fish much like the mutants in the
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mouse show very mild vascular defects if
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any only about 5% of mutant show this
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defect but as you can see here again
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from the original data using the
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anti-sense technology we have both
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severe vascular defects vascular tube
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defects that is so defects in vascular
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Genesis as well as defect in
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androgenesis that leads to the spring of
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new vessels so essentially we have as
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been seen observed for several other
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genes now we have profound phenotypic
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differences between the egfl7 mutants
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and the egfl7 morphins and one can think
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about number of simple or trivial
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explanations why that would be it's
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possible that the mutant re we had made
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was a hypomorph and it's also possible
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that the Morino that had been used for
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these studies were inducing of Target
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effects and this is the phenotypes that
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essentially people had been looking at
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it's also possible that there was a more
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interesting observation and so we
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decided to look further and try and
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understand the discrepancy between the
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mutant and the morphin
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phenotype so essentially uh the first
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question then is is the mutation a noal
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and you might think this is a simple
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question to answer but in fact the
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genome has come up with many different
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ways to bypass mutations especially
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mutations that lead to stop codons
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premature stop codons in the five Prime
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of the gene so for example it's been
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observed now several times that
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Downstream atgs or even non atg codons
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can be used for initiation or
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translation we can uh We've also
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observed and I'll show you in a minute
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Exxon skipping and then in terms of
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secreted proteins like
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egfl7 certainly one can also Imagine
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scenarios where unconventional secretion
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pathways are used for truncated proteins
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for example
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I may show you an example of excellent
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skipping again that's been observed
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several times in the field and in this
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case we made mutations in Exon 2 and as
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you can see again here these are now two
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different mutations there's a mutation
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one and mutation two and in the mutant
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you can see essentially right here that
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there's a smaller band that's also
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present in zygus and this band comes
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from the skipping of hexon 2 as you can
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see there so essentially as I said many
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ways by for by which the genome can
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circumvent what looks to be severe
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lesions so in terms of our lesion our
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Delta 4 mutation specifically what we
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did is first look at the RNA levels as
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shown here and you can see there's a
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reduction in the Delta 4 Al compared to
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Wild type or the Delta 3 Al so there's a
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about 50% reduction in a tript level
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possibly through nonsense Med Decay if
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you look at the protein we express both
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the wild type and the mutant protein in
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cells and as it is a secreted protein
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you can see that most of the wild type
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protein is present in the medium if you
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look at the mutant protein you can see
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it has a reduction in the level of
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expression but you can see very little
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protein in fact secret so these two data
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together suggest that this Al this Delta
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four Al that we are generating it could
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in fact be a severe
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Le how about the second question what
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about of Target effects caused by
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Molinos now in order to do this we're
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going to be injecting the Molino into
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this mutant Al that we made and
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essentially the reasoning here is that
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if this Al this m al is a null the M any
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additional phenotypes that's seen from
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Morino injection should by definition be
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enough Target effect so essentially
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before we do that before we inject this
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Morino into the egfl7 mutants we want
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first to introduce a MC tag in the egfl7
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locus Again by Gene editing following
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cleavage by tailin and this is to allow
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us to look at the efficiency of De
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Morino at different Doses and so by
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Western blood analysis then after
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injecting one nanogram of this Morino
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into these transgenic embryos one can
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see in fact there's about an 80%
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reduction of protein levels egfl protein
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levels using one nanogram Morino we
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chose this dose of one nanogram because
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if you inject higher doses as you can
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see here you essentially induce the
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expression of
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p53 which has been a reported to be
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indicative of an of Target effect from
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morpholino injections and so essentially
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one nanogram does not cause p53
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induction but uh two will and so we
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stuck with one
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nanogram so essentially now we're ready
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for the experiment so we're going to be
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injecting this EF mosino into efl 7
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mutants in the following manner we're
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going to be Crossing heads injecting one
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anram of
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lopeno and then taking 32 affected
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embryos and genotype them and so let's
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first look at the various
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scenarios that and the outcomes of what
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would predict so essentially if the
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mutant Al is not null then the mutant
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embryo should be more sensitive than the
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wild type to the Mineo injections let's
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say for example there's 20% Gene
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function left you inject the moino one
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nanogram the mutant embryo should be
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more
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sensitive if the mutant is null and the
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M phenotype is due to of Target effects
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then essentially the genotype of the
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embryo should not matter the mutant and
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W should be equally sensitive to
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morpholino
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injections however if the mutant is now
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the Morino phenotype is not due to of
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Target effects then the muton embryo
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should be less sensitive than the wild
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type to the Molino injections and this
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is exactly in fact what we SE so we as I
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said genotype 32 affected
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embryos and we would expect through
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Melia segregation eight of them to be
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mutants but in fact we only found three
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of them here shown in this red
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curves and so this indicates that in
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fact the je7 mutants are less sensitive
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they are somewhat protective protected
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to the jeffr 7 Morino and so in fact
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these are the data now different
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experiments in the control experiment
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you can see Mandarian segregation of the
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various genotypes but when you inject
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the Morino you can see that out of 32
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injected embryos fewer than eight of
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them are in fact showing a phenotype and
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you can of course then look at this
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retrospectively after you've genotyped
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the embryos go back to the pictures that
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you took and this is for example here a
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wild type embryo that was injected with
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one nanogram on the Molino in this case
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a mutant embryo you can see that the
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mutant embryo does not show any
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androgenesis phenotype we're looking
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here at the formation of these vessels
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here in the trunk that form through
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androgenesis the So-Cal inic vessels and
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you can see clear phenotype in the wild
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type but not in the mutant
00:16:30
so essentially we have this situation
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where the mutants do not show a severe
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phenotype but if you inhibit translation
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you see a severe phenotype this is by
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now using these Morino anti-sense what
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about now if you inhibit transcription
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what will you see and the way we did
00:16:47
this inhibiting transcription was take
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advantage again of a recently developed
00:16:52
technique called crisper interference
00:16:55
and we're using now a Dead version of
00:16:57
cast 9 that one that doesn't have NE
00:16:58
play activity to block
00:17:01
transcription and so here are the
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experiments first to show that in fact
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we can block transcription to about 50%
00:17:08
level and so here are guides used
00:17:12
against both the template and
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non-template strands and in fact when
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you use this approach you can phenocopy
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the morphin induced phenotypes or the
00:17:23
morphin like phenotype so again these
00:17:25
are again control and to experimental
00:17:28
and you can see defects in the inic
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vessels as shown
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here so what we have is a situation
00:17:36
where the mutants don't show a phenotype
00:17:38
but if you use morphon to block
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translation or you use this crisper
00:17:42
interference to block transcription you
00:17:44
see a severe vascular
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defects so essentially the hypothesis
00:17:50
then became one of Gene compensation and
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to use a classical example one from the
00:17:56
muscular distrophy field when mount
00:17:58
house mutants were made for the drine
00:18:01
gene utrine Gene was upregulated and so
00:18:04
one needs to make the double mutant to
00:18:06
see the kind of phenotype that the Duan
00:18:09
patients exhibit and so the hypothesis
00:18:12
then in our case was that there was in
00:18:14
fact the activation of a network a
00:18:17
compensatory Network that would buffer
00:18:19
against the ous mutations and this
00:18:21
compensation was present in mutant
00:18:23
embryos but not in Morphin or in crisper
00:18:26
eye injected embryos and so we use and
00:18:29
proteomics and transcriptomics to test
00:18:31
this hypothesis and let's look first at
00:18:34
the proteomics so we're comparing while
00:18:37
type mutant and morphins and what we
00:18:40
found is essentially a single protein
00:18:43
this is now comparing mutant to Wild
00:18:45
type we found a single protein emine 3A
00:18:48
that's upregulated in the mutant
00:18:51
compared to the wild type but
00:18:53
interestingly this emine 3 3A was not
00:18:57
significantly a regulated
00:18:59
in the morphant compared to the wild
00:19:03
type looking at the RNA levels we found
00:19:07
in fact not only emine 3A but other
00:19:09
family members including mine 3B and
00:19:12
emine 2A and you can see again that they
00:19:15
are
00:19:16
upregulated in the mutant compared to
00:19:19
the wild type but not in the morphant
00:19:22
similarly when we use crisper eye we do
00:19:25
not see we did not see a regulation of
00:19:27
these genes me 3A 3 a 3B and
00:19:31
2A what are these emaline genes uh we
00:19:35
know in fact that like egfl7 emalin are
00:19:38
negative Regulators of elastogenesis and
00:19:41
one of the main and functional domain of
00:19:44
egfl7 shown here in yellow is in fact
00:19:47
Emy domain and this name Emy comes in
00:19:50
fact from the amaline genes now are
00:19:54
these genes the upregulation of these
00:19:56
emine genes in fact important
00:19:58
functionally
00:19:59
and as it can it explain you find the
00:20:01
lack of phenotypes in egfl7 mutants and
00:20:04
the way we addressed this question is by
00:20:08
essentially making egfl7 Morphin and
00:20:11
then rescuing them with wild type as
00:20:13
well as mutant egfr 7 as well as Amin 2
00:20:17
and mine 3 and as you can see here again
00:20:20
this is now the number of effect in
00:20:23
green we're looking at the
00:20:24
phenotypically normal embryos which are
00:20:27
few and when you inject the efr 7 Morino
00:20:31
when you come in with E7 RNA for rescue
00:20:34
you can see that this frequent number
00:20:37
frequency
00:20:38
increases if you use a mutant version of
00:20:41
efl 7 you fail to rescue and again much
00:20:44
like w type ej7 you can partially rescue
00:20:48
the ej7 morph and phot type by using
00:20:51
this MN 2 and M3
00:20:54
genes this compensation phenomenon that
00:20:57
we observed in zebra fish this
00:20:59
difference between knockout and
00:21:02
knockdown or morphant and mutant mutant
00:21:05
and morphant embryos is also observed in
00:21:08
yeast there was a recent study from
00:21:10
Orion wiers lab where they looked at the
00:21:13
B one gene if you look at the mutation
00:21:16
in bem one as you can see here
00:21:19
essentially there causes a very minor
00:21:21
phenotypes now but now if you use
00:21:23
optogenetics to drive this protein away
00:21:25
from its side of action then you see now
00:21:28
a severe phenotypes including cell cycle
00:21:30
arrest and cell
00:21:32
liis so to summarize and uh essentially
00:21:35
provide some outlook on these studies
00:21:38
clearly there are some moros that phoc
00:21:41
cop imitations at least at the
00:21:42
morphological level for example kyot
00:21:45
tropine te that we use extensively in
00:21:47
part two to block contraction of the
00:21:50
heart there are other morpholinos that
00:21:53
do not phop imitations so there are
00:21:56
possible a number of possible
00:21:57
explanations including the fact that
00:22:01
mutos could be hypermorphic and that's
00:22:03
certainly could be the case for many
00:22:05
mutations that were induced in the five
00:22:07
pandum genes there are going to be moros
00:22:11
that in fact do cause a number of of
00:22:14
Target effects even if used at a
00:22:17
relative low dose and then in some cases
00:22:21
for example as we just observed with
00:22:24
egfr 7s we're going to have compensation
00:22:27
in the mutants but but not in the
00:22:30
morphin what about morphos how now with
00:22:35
this in mind and with the ability to
00:22:37
essentially then mutate any Gene using
00:22:41
Talon's crisal 9 how should we think
00:22:44
about using
00:22:46
Molinos and the argument would be that
00:22:48
in fact to find a Molino that causes no
00:22:52
of Target effects and probably the best
00:22:54
way to do this is to find a dose and a
00:22:57
sequence of muina that has effect in the
00:22:59
corresponding nor mutant embryos or
00:23:01
maybe even better yet in embryos that
00:23:03
are lacking The morpholino Binding
00:23:06
site since we do see differences between
00:23:09
the morphant and mutant phenotypes a
00:23:11
question of course arises as to which of
00:23:14
these phenotypes is the real phenotype
00:23:16
and which tool to use and we would argue
00:23:18
that of course both of these tools
00:23:20
mutation approach as well as the mine or
00:23:23
anti- sensor Pro should be used even if
00:23:25
they give you different answers both of
00:23:27
these answers could in fact be correct
00:23:29
especially if the anti-sense reagent has
00:23:32
been validated
00:23:33
previously now the zfish is particularly
00:23:37
well suited to do this kind of work it
00:23:39
is to compare mutant and morphan
00:23:41
phenotypes and one way we are thinking
00:23:44
of using it is essentially to identify
00:23:47
members of the network so for example in
00:23:49
this context the context of the work I
00:23:51
just described you might think of MN as
00:23:54
being part of a network with egfr 7 and
00:23:57
MN could in fact because it can at least
00:24:00
partially compensate for the lack of
00:24:02
agfr 7 it can be by definition seen as a
00:24:05
modifier Gene and so essentially the
00:24:07
idea now is to take genes implicated in
00:24:10
vascular development vascular biology
00:24:12
and see if theem Morphin and mutants for
00:24:14
these genes show different phenotypes
00:24:17
and if they do can we identify the
00:24:18
compensating genes and thereby identify
00:24:21
the modify
00:24:23
genes now mechanistically there are a
00:24:25
number of course of interesting
00:24:26
questions including what what are the
00:24:29
mechanisms of this transcriptional
00:24:30
regulation so in the efl 7 mutants how
00:24:34
does mln transcription get upregulated
00:24:38
what is the trigger for this
00:24:39
upregulation what are the mechanisms
00:24:41
between the trigger itself and this uh
00:24:44
transcriptional AB regulation so this is
00:24:47
some of the ongoing work now in the lab
00:24:50
and uh let me just give you a few SL
00:24:52
show you a few slides about what we're
00:24:54
doing not only in zebra fish but also in
00:24:57
miman cells
00:24:59
we now have a number identify a number
00:25:01
of genes where essentially we see this
00:25:05
RNA level of regulation in the Power log
00:25:09
the non-mutated Power log and in four of
00:25:12
these cases we've also seen that the
00:25:13
hetus embryos as well as the mutant
00:25:15
embryos show in fact this increased mRNA
00:25:18
levels so for example I've told you
00:25:21
about in this case earlier about egfr 7
00:25:24
and the Abul by of Emin we've also
00:25:28
looked at VF AA mutants and we see a
00:25:31
regulation of VF
00:25:34
AB if you looked at the hetus embryo
00:25:37
again for example looking at egfl7 right
00:25:41
here you can see that the atrous embryo
00:25:44
show an intermediate level of up
00:25:46
regulation compared to the mutants and
00:25:48
of course here is the W type and
00:25:50
similarly vfa also atus embryos also sh
00:25:55
Show this intermediate level of ab
00:25:56
regulation
00:25:58
now of course we've also identified and
00:26:00
seen observed genes that when mutated do
00:26:03
not cause the ab regulation of the
00:26:05
paralog and some of these cases are
00:26:08
shown here and so with that I'll thank
00:26:11
the acknowledge the people who've been
00:26:14
driving these projects including the
00:26:16
original paper rosi contus at all and
00:26:20
also mohamad's work is now working and
00:26:23
to essentially look further into this
00:26:25
phenomenon of compensation using zebra
00:26:28
fish mutants but also using miman sound
00:26:31
lines where we observe similar
00:26:33
compensation phenomenon trying to see if
00:26:35
we can get into mechanisms again not
00:26:37
only to identify the trigger but also
00:26:40
the mechanism between the trigger and
00:26:43
the transcription AB regulation and I
00:26:44
also want to thank the funding buddies
00:26:47
uh who supporting this work thank
00:26:57
you for
