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 β-globin gene with the sickle mutation (βSH) 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 α-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 α-globin and β-globin genes, and that’s what we will cover today. So…how do we “get rid of” the endogenous mouse α-globin and β-globin genes? Just like making transgenic mice this involves some manipulations of the mouse embryo…but this is a much more complex process, and some background about the preimplantation mouse embryo is needed. For about 4-5 days after fertilization, the mouse embryo is 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 pre-implantation embryos (e.g., embryos destined to PROBLEM: These mice still do not have RBCs that sickle very well. The mouse still has mouse α and β globin molecules and their presence is enough to prevent the human hemoglobins from forming fibers, in much the same way that humans heterozygous for the sickle mutation do not normally have RBCs that sickle.SOLUTION: Need to get rid of the endogenous mouse α and βglobin genes by targeted homologous recombination to generate “Knock-out” miceβMβMαMαMαMαMβHSβHSβHSβHSβHSβHSHHαHHHαHPROBLEM: These mice still do not have RBCs that sickle very well. The mouse still has mouse α and β globin molecules and their presence is enough to prevent the human hemoglobins from forming fibers, in much the same way that humans heterozygous for the sickle mutation do not normally have RBCs that sickle.SOLUTION: Need to get rid of the endogenous mouse α and βglobin genes by targeted homologous recombination to generate “Knock-out” miceβMβMαMαMαMαMβHSβHSβHSβHSβHSβHSHHαHHHαHβMβMαMαMαMαMβHSβHSβHSβHSβHSβHSHHαHHHαHHHαHHHαHproduce 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 knock-out technology. Early findings revealed that the preimplantationmouse embryo is remarkably malleable, and that cells in the thepreimplantationembryo are TOTIPOTENTEarly findings revealed that the preimplantationmouse embryo is remarkably malleable, and that cells in the thepreimplantationembryo are TOTIPOTENT 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 Tn7TRTn7TR lacZ URA3 tet Tn7TRTn7TR lacZ URA3 tet Tn7TRTn7TR lacZ URA3 tet Tn7TRIn yeastYeast genomic DNAIn yeast homologous recombination to replace an endogenous gene with the transfected DNA fragment occurs >90% of the timeIn 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 recombinationTn7TR lacZ URA3 tet Tn7TRTn7TR lacZ URA3 tet Tn7TRTn7TR lacZ URA3 tet Tn7TRTn7TR lacZ URA3 tet Tn7TRIn yeastYeast genomic DNATn7TR lacZ URA3 tet Tn7TRTn7TR lacZ URA3 tet Tn7TRTn7TR lacZ URA3 tet Tn7TRTn7TR lacZ URA3 tet Tn7TRIn yeastYeast genomic DNAIn yeast homologous recombination to replace an endogenous gene with the transfected DNA fragment occurs >90% of the timeIn 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. 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
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