Video instructions and help with filling out and completing Where Form 5495 Compensation

Instructions and Help about Where Form 5495 Compensation

Hello my name is DDS tinium directed the Max Planck Institute for research in Bad Nauheim Germany and today this third part we're going to be talking about the phenomenon of genetic compensation in the context of a scary development in the zebrafish embryo historically gene function in zebrafish has been studied initially through a full genetic approach mainly for utilizing the genome introducing mutations randomly and then doing phenotypic screening more recently morpholino zant essentially goes had been used to knockdown gene function and then more recently just a few years ago using zinc finger nucleases as well as talents and Chris Burke has nine technology mutations have been introduced in specific genes to study not only genes that were studied previously using oph lie notes but also other genes and the studies that I'll be telling you about today or essentially inspired by the fact that looking at specific genes that have been studied was using more funny nose as well as reversed genetic techniques it became apparent that in many cases the phenotypes induced by mutations or much milder than those induced by the morpholino technology and so essentially just to give you a little background Mourinho's are as you can see here modifying all egos they're highly stable they bind RNA there used to rock neither translation or splicing and so the question then is why mutant phenotypes are in sight of a milder than Tysons phenotypes so mutant phenotypes versus anti-fans phenotypes I will also be using the word more front for morpholino induced so we'll be talking about mutant versus morph and phenotype and mutants often refer to as knockouts and anti sensors knockdowns so let's understand by some history of the anti sense approach anti sense technology and more than thirty years ago people working in developmental biology or using or started using anti sense RNA - essentially blocked in function it is it was in the context of frog embryo as well as the fly embryo but this period was fairly short-lived probably because people were concerned about of target effects and so people moved to overexpressing neither wild-type or dominant negative versions of genes or proteins and essentially for example in this case dominant negative accident receptor certainly was known and to interfere with other proteins besides the actinin receptor and yet approaches such as this gave us important insights into developmental processes and so it's important to realize that while no reagent is perfect certainly these reagents can be used to make important insights into biological processes and you know zebrafish defense technology using the more funny nose was introduced in 2000 at the same time as it was introduced in the Frog and a few years later a number of guidelines were written up essentially to try and asbestos people could essentially control for these money no studies and specifically trying to avoid or and recognize of target effects and so essentially with this in mind and as I said as the various reverse genetic techniques became available people started saying essentially important differences between the mutation induced phenotypes and the morpholino or anti science in Ticino types and this was further emphasized by the larger study from nathan Larson's lab where they essentially looked at a large number of genes and again found a poor correlation between morpholino induced and mutant phenotypes and zebrafish and so essentially this is not specific I should mention to the zebrafish field in fact if you now look at an entire science work in the mouse is now using transgenesis to drive anti sense try transcript essentially again you see a more severe phenotypes from the anti sense approach then from the mutation approach and so it's clearly a question that spans beyond or goes beyond just using molinos and pori applies to all anti sense work so we decided to revisit this issue in detail and we picked this gene called EGFR 7 for a number of reasons but mostly because in three different settings using the anti sense approach this gene and been implicated in there playing an important role in vascular development this was in zebrafish frog as well as in human and a theatre cells and yet in the masked mutant there was no phenotype no discernable phenotype just give you a little background where this gene in encodes in ECM protein is expressed mostly by endothelial cells and it's apparently expressed by tumor cells in human cancers and for this reason where's the a drug target in turn on taking flight had a clinical trial for one of the human eyes I'm going to call anybody against this protein so as I mentioned in the diba fish where the first work was done on this gene when you knock down EGFR 7 using more phone you know you see severe defects in number of processes including vascular to formation and you also get the pericardial edema indicative of a failing heart similar phenotype was seen in the frog embryo again using an anti sensor approach and also well as in human ES cells human and deceive yourself and as I mentioned here in the contrast to these studies to these findings the mouse mutant was in fact phenotypically normal now this was a little complicated initially buying a fight that there is a indeed if our seven loka's as you can see here there is the my cornea my carney 126 embedded in this locus and this mic RNA is also expressed in a few cells and so in fact the original Nutan that was made deleted this my colony as well and they sled to the appearance of vascular phenotypes but in fact when a specific knock out big mutations were made either in the micron a or eg t47 gene itself it was realized in fact the micro RNA was the one responsible for the phenotype seen in the original mutant so essentially the