Lecture 24 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 introduced as a transgene in mice in the hope that it would cause the as discussed how the human B-globin gene with the sickle mutation (Bsh)was 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 a-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 a-globin and B-globin genes, and that's what we will cover today. BUT, before then, I want to share with you some great questions that i got after the last lecture, and some responses to those questions. Great Questions from students after the last lecture now it didn't integrate into an 需温 PROBLEM: These mice still do not have RBCs that sickle very well The mouse still has mouse a and B globin molecules and their presence is enough to prevert the human hemoglobins from forming fibers, in much the same way that humans heterozygous for the hy didn't the human sickle mutation do not normally have RBcs that sick SOLUTION Need to get rid of the endogenous mouse a and B globin genes by targeted homologous recombination to generate Figure by MIT OCW. K So.how do we "get rid of"the endogenous mouse a-globin and B-globin genes? Just like making transgenic mice this involves some manipulations of the mouse embryo.but this is a much more Images removed due to copyright reasons. 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 toLecture 24 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 (βS H) 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. BUT, before then, I want to share with you some great questions that I got after the last lecture, and some responses to those questions. Great Questions from students after the last lecture • How do you know it didn’t integrate into an important gene? • Can’t the phenotype (if you get one) be because of the disruption of an endogenous gene? • How do you know that the human globin proteins were expressed? • Why didn’t the human βS-globin gene recombine with the mouse β-globin gene? • Could one inject the w.t. human β-globin gene into a human embryo to correct the deficiency? Great Questions from students after the last lecture • How do you know it didn’t integrate into an important gene? • Can’t the phenotype (if you get one) be because of the disruption of an endogenous gene? • How do you know that the human globin proteins were expressed? • How do you know it didn’t integrate into an important gene? • Can’t the phenotype (if you get one) be because of the disruption of an endogenous gene? • How do you know that the human globin proteins were expressed? • Why didn’t the human βS-globin gene recombine with the mouse β-globin gene? • Could one inject the w.t. human β-globin gene into a human embryo to correct the deficiency? 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 β globin genes by targeted homologous recombination to generate “Knock-out” mice βM βM αM αM αM αM βH S βH S αH αH 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 β globin genes by targeted homologous recombination to generate “Knock-out” mice βM βM αM αM αM αM βH S βH S αH αH βM βM αM αM αM αM βH S βH S αH αH 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 Transfer injected eggs into foster mother. Inject foreign DNA into one of the pronuclei Fertilized mouse egg prior to fusion of male and female pronuclei Pronuclei About 10-30% of offspring will contain foreign DNA in chromosomes of all their tissues and germ line Breed mice expressing foreign DNA to propagate DNA in germ line Images removed due to copyright reasons. Figure by MIT OCW