7 03 2005 Lecture 25 Transgenes and Gene Targeting in Mice II In the last lecture we discussed sickle cell disease SCD in humans and I told you the first part of a rather long but interesting story describing how a mouse model for this human disease has been generated I only got half way through the story we will cover the rest today In the last lecture we discussed how the human globin gene with the sickle mutation SH was introduced as a transgene in mice in the hope that it would cause the precipitation of hemoglobin and the sickling of mouse red blood cells RBCs had this happened this would have generated an animal model for SCD If you recall the transgenic mouse did not have sickling RBCs and to try to fix this the human globin gene was also introduced into the mouse genome but still the doubly transgenic mouse did not have sickling RBCs The solution to this was to inactivate the endogenous mouse globin and globin genes and that s what we will cover today H SH So how do we get rid of the endogenous mouse globin and SH H globin genes Just like making M M M transgenic mice this involves some manipulations of the mouse PROBLEM These mice still do not have RBCs that sickle very well embryo but this is a much more The mouse still has mouse and globin molecules and their complex process and some presence is enough to prevent the human hemoglobins from forming fibers in much the same way that humans heterozygous for the background about the sickle mutation do not normally have RBCs that sickle preimplantation mouse embryo is needed For about 4 5 days after SOLUTION Need to get rid of the endogenous mouse and fertilization the mouse embryo is globin genes by targeted homologous recombination to generate Knock out mice freefloating and therefore accessible and all of the cells that will eventually form the mouse remain totipotent meaning that they have the potential to differentaite into any and every mouse cell type This has been shown in various dramatic ways For instance if the four cell embryo is dissected and each cell implanted into a different foster mother four identical mice will be born More interestingly if cells from two genetically different preimplantation embryos e g embryos destined to M M M Early findings revealed that the preimplantation mouse embryo is remarkably malleable and that cells in the the preimplantation embryo are TOTIPOTENT produce mice with different fur colors are simply mixed together they are sticky and implanted into a foster mother a single chimeric mouse will be born Essentially the two types of totipotent cells mix together and produce an animal that has a micture two types of cells in its body This animal has four genetic parents The ability of these genetically different totipotent cells to mix together in the preimplantation embryo is crucial for the mouse gene knockout technology In order to make a directed genetic change in a specific mouse gene we exploit homologous recombination just as we have discussed for E coli and S cerevisiae However this is much harder to do in mammalian cells than bacteria and yeast In yeast when a linear In yeast DNA duplex is introduced into the cell about 90 of the time that that DNA is integrated into the yeast genome it is done Yeast genomic DNA by the homologous recombination machinery such that incoming DNA In yeast homologous recombination to replace an endogenous gene with the transfected DNA fragment fragment is swapped for the endogenous occurs 90 of the time gene In mammalian cells the DNA that is In mammalian cells such homologous recombination between genome and transfected DNA fragment is very integrated into the genome is almost always rare 0 01 of the time at a non homologous site and the Have to have clever selection schemes to get the rare cells frequency of homologous replacement of an that integrated a transfected DNA fragment by targeted homologous recombination endogenous sequence is about 10 3 to 10 5 What this means is that we have to allow thousands of integration events to take place and to be able to identify the integration event we want namely an integration even that took place by homologous recombination Tn7TR lacZ URA3 tet Tn7TR The first crucial development for this technology was being able to grow the totipotent cells from preimplantation embryos in culture in the lab these are called mouse embryonic stem cells ES cells the crucial development was to devise a clever way to select integrated a DNA construct by homologous recombination Cells from the inner cells mass of a preimplantation embryo at the blastocyst stage could be removed and cultured in the lab without the cells losing their totipotency i e even after being cultured in the lab for many years these cells can still be introduced back into a preimplantation embryo and go on to make all the tissues of a mouse What this means is that the cells can be genetically manipulated whilst in culture and then put back into a mouse preimplantation embryo Preimplantation blastocyst from an embryo that would produce a mouse with GREY FUR Specifically replace your gene of interest or globin genes with a mutated version of that gene in cultured ES cells Can remove totipotent EMRYONIC STEM CELLS ES cells and culture in vitro Select for the genetically altered cells you want Targeting Construct NeoR TKHSV Select for the NeoR gene and against the TKHSV gene The only cells to survive have undergone a targeted homologous recombination event at the gene of interest Select for the genetically altered cells you want Now you inject the genetically modified ES cells originally from a blastocyst for a mouse with GREY FUR and inject into a new blastocyst that would normally give rise to a mouse with WHITE FUR The blastocyst now containing two types of totipotent embryonic stem cells is implanted into a foster mother she will give birth the chimeric offspring Essentially once you have identified mouse ES cells originally from a grey furred mouse that have been genetically altered the way you wish these cells can be used to generate a living animal that contains descendents from these totipotent ES cells Lets see how you get from there to a mouse in which every cell contains that genetic alteration Foster Mom Some mice are Chimeric The goal is to have the GERM CELLS sperm and eggs derived from the genetically modified ES cells if so all the offspring would have GREY FUR when mated with a white mouse grey fur is a dominant trait Since the grey ES
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