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 pre-implantation embryos (e. g. embryos destined to 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 Early findings Essentially the two types of revealed that totipotent cells mix together and the produce an animal that has a preimplantation micture two types of cells in its Images removed due to mouse embryo body. This animal has four geneti copyright reasons is remarkably parents ! malleable, and The ability of these genetically that cells in the different totipotent cells to mix the together in the preimplantation preimplantation embryo is crucial for the mouse TOTIPOTENT/ gene knock-out technology Se embryo are 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 DNa duplex is introduced into the cell about 90% of the time that that dna is integrated into the yeast genome it is done by the homologous recombination Yeast genomic DNA machinery such that incoming dNA recombination to replace an fragment is swapped for the endogenous endogenous gene with the transfected DNA fragment occurs 90% of the time gene. In mammalian cells the dna that is In mammalian cells such homologous recombination integrated into the genome is almost always between genome and transfected DNA fragment is very at a non-homologous site, and the rare(0.01% of the time 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 endogenous sequence is about 10 to 10 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 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 recombinationdifferentaite 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 pre-implantation embryos (e.g., embryos destined to 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!! Early findings revealed that the preimplantation mouse embryo is remarkably malleable, and that cells in the the preimplantation embryo are TOTIPOTENT Early findings revealed that the preimplantation mouse embryo is remarkably malleable, and that cells in the the preimplantation embryo are TOTIPOTENT The ability of these genetically different totipotent cells to mix together in the preimplantation embryo is crucial for the mouse gene knock-out 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 DNA duplex is introduced into the cell, about 90% of the time that that DNA is integrated into the yeast genome it is done by the homologous recombination machinery such that incoming DNA fragment is swapped for the endogenous gene. In mammalian cells the DNA that is integrated into the genome is almost always at a non-homologous site, and the frequency of homologous replacement of an 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 In yeast Yeast genomic DNA In yeast homologous recombination to replace an endogenous gene with the transfected DNA fragment occurs >90% of the time In mammalian cells such homologous recombination between genome and transfected DNA fragment is very rare (<0.01% of the time) Have to have clever selection schemes to get the rare cells that integrated a transfected DNA fragment by targeted homologous recombination Tn7TR lacZ URA3 tet Tn7TR In yeast Yeast genomic DNA Tn7TR lacZ URA3 tet Tn7TR In yeast Yeast genomic DNA In yeast homologous recombination to replace an endogenous gene with the transfected DNA fragment occurs >90% of the time In mammalian cells such homologous recombination between genome and transfected DNA fragment is very rare (<0.01% of the time) Have to have clever selection schemes to get the rare cells that integrated a transfected DNA fragment by targeted homologous recombination 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. Images removed due to copyright reasons