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 discussed how the human B-globin gene with the sickle mutation(BsH)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 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. CH Great Questions from students after BM the last lecture BM Pronudei ★ a电xgeagtddnritegnoteitoam 6eam地.iteP+nou PROBLEM:These mice still do not have RBCs that sickle very well. gene? 6acaaa9s The mouse still has mouse a and B globin molecules and their f© presence is enough to prevent the human hemoglobins from forming fibers,in much the same way that humans heterozygous for the ○⊙灯● sickle mutation do not normally have RBCs that sickle gee2的8g器5ene7 Could one inject the w.t.human B-globin SOLUTION Need to get rid of the endogenous mouse a and B emytocthe globin genes by targeted homologous recombination to generate "Knock-out"mice Figure by MITOCW..长O长O长O 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 to
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 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
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 mouse embryo Images removed due to body.This animal has four genetic is remarkably copyright reasons. 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 embryo are TOTIPOTENT 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, In yeast about 90%of the time thatthat 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 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) frequency of homologous replacement of an Have to have clever selection schemes to get the rare cells that integrated a transfected DNA fragment by targeted endogenous sequence is about 103 to 105 homologous recombination 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 recombination
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. 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 (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
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 Specifically replace your gene neo mouse with a n 如== gene in ultured ES cells Gene X replacement construct Nonhomologous Homologous recombination recombination 是上 位归 ES-cell DNA ES-cell DNA Othe Gene X aeeoan - = No mutation in gene X Mutation in gene X Select for the genetically altered Cells are resistant to G-418 Cells are resistant to G-418 cells you want out sensitive to gancidovir and ganciclovir (ES cells)and culture in vitro Formation of ES Cells Carrying a Knockout Mutation Targeting Construct Now you inject the genetically modified an the s ee ES cells (originally from a blastocyst for a new bia hat8沿A6re rise to a mouse with WHITE FUR The only cells to survive have undergone a targeted homologous recombination The blastocyst,now containing two types of totipotent embryonic stem event at the gene of interest cells,is implanted into a foster mother; she will give birth to the chimeric offspring Select fot the genetically altered cells you want Figures by MIT OCW. 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
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!! 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. Preimplantation blastocyst from an embryo that would produce a mouse with GREY FUR Can remove totipotent EMBRYONIC STEM CELLS (ES cells) and culture in vitro Select for the genetically altered cells you want Construct Specifically replace your gene of interest (α or β-globin genes) with a mutated version of that gene in cultured ES cells Gene X replacement construct neor tkHSV Homologous recombination Gene-targeted insertion Gene X Mutation in gene X Cells are resistant to G-418 and ganciclovir Formation of ES Cells Carrying a Knockout Mutation ES cells ES-cell DNA Random insertion Other genes No mutation in gene X Cells are resistant to G-418 but sensitive to ganciclovir ES cells ES-cell DNA Nonhomologous recombination Targeting Construct 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 fot the genetically altered cells you want NeoR TKHSV 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 to the chimeric offspring Figures by MIT OCW
The goal is to have the Heterozygous for Heterozygous for the knocked out the knocked out Foster GERM CELLS (sperm and eggs)derived from the gene gene genetically modified Es cells;if so all the offspring would have GREy FUR when mated with a white mouse grey CM+/- aM+/- Some mice are Chimeric fur is a dominant trait M+/+ aM+/- a.M -/ Since the "grey"ES cells were heterozygous for the KOd 25% 50% 25% gene,only half the sperm have the KO gene.so 50%of the grey offspring are Homozygous mutant heterozygous for the KO. mice...Viable?? The blastocyts implanted into the foster mother will produce animals with varying contributions from the "white fur ES cells"and the "grey fur ES cells", the latter having been genetically manipulated to have an altered gene,e.g.,a mutated a-globin gene.The crucial step is that the gonads be derived from the genetically altered "grey fur ES cells",because then the genetic alteration can be passed on to an offspring (which will have grey fur)in which every cell carries the genetic alteration.These offspring can then be crossed to generate a mouse that is homozygous for the altered gene.This can be done for generating mice with deletion mutations in the a-globin gene and then again for deletion mutations in the B-globin gene. SPERM 4p4 upu auprr ampu This is essentially an AaBb X AaBb cross +++/+ +/++/ +/++ +/-+/ where the A and B cenes lie on different chromosomes and are QMEBM- +/++ ++-4 +h+ +/ therefore unlinked. k 立 尚 The Human transgenes 部 aM-β+ +1-+/+ +/-+/ +丹 -+ are homozygous in both parents and so will be present in all offspring. +-+ -+ 1/16 offspring have the desired genotype X NeoR NeoR NeoR There are many different mating schemes that one could use to generate mice that are homozygous for deletions in both the mouse a-globin gene and the
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 Foster Mom Since the “grey” ES cells were heterozygous for the KO’d gene, only half the sperm have the KO gene, so 50% of the grey offspring are heterozygous for the KO. 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 Foster Mom Since the “grey” ES cells were heterozygous for the KO’d gene, only half the sperm have the KO gene, so 50% of the grey offspring are heterozygous for the KO. Some mice are Chimeric Heterozygous for the knocked out gene Heterozygous for the knocked out gene αM +/- αM +/- αM +/+ αM +/- αM -/- 25% 50% 25% Homozygous mutant mice… Viable?? Heterozygous for the knocked out gene Heterozygous for the knocked out gene αM +/- αM +/- αM +/+ αM +/- αM -/- 25% 50% 25% Homozygous mutant mice… Viable?? The blastocyts implanted into the foster mother will produce animals with varying contributions from the “white fur ES cells” and the “grey fur ES cells”, the latter having been genetically manipulated to have an altered gene, e.g., a mutated α-globin gene. The crucial step is that the gonads be derived from the genetically altered “grey fur ES cells”, because then the genetic alteration can be passed on to an offspring (which will have grey fur) in which every cell carries the genetic alteration. These offspring can then be crossed to generate a mouse that is homozygous for the altered gene. This can be done for generating mice with deletion mutations in the α-globin gene and then again for deletion mutations in the β-globin gene. +/+ +/+ +/+ +/- +/- +/+ +/- +/- +/+ +/- +/+ -/- +/- +/- +/- -/- +/- +/+ +/- +/- +/- +/- -/- +/+ +/- -/- -/- +/- -/- +/- -/- -/- αΜ+βΜ+ αΜ+βΜ+ αΜ+βΜ− αΜ+βΜ− αΜ−βΜ+ αΜ−βΜ+ αΜ−βΜ− αΜ−βΜ− EGGS SPERM This is essentially an AaBb X AaBb cross where the A and B genes lie on different chromosomes and are therefore unlinked. The Human transgenes are homozygous in both parents and so will be present in all offspring. 1/16 offspri offspring have the desired genotype βH S βH S αH αH NeoR NeoR NeoR NeoR βH S βH S αH αH NeoR NeoR NeoR NeoR +/+ +/+ +/+ +/- +/- +/+ +/- +/- +/+ +/- +/+ -/- +/- +/- +/- -/- +/- +/+ +/- +/- +/- +/- -/- +/+ +/- -/- -/- +/- -/- +/- -/- -/- +/+ +/+ +/+ +/- +/- +/+ +/- +/- +/+ +/- +/+ -/- +/- +/- +/- -/- +/- +/+ +/- +/- +/- +/- -/- +/+ +/- -/- -/- +/- -/- +/- -/- -/- αΜ+βΜ+ αΜ+βΜ+ αΜ+βΜ− αΜ+βΜ− αΜ−βΜ+ αΜ−βΜ+ αΜ−βΜ− αΜ−βΜ− EGGS SPERM This is essentially an AaBb X AaBb cross where the A and B genes lie on different chromosomes and are therefore unlinked. The Human transgenes are homozygous in both parents and so will be present in all offspring. 1/16 offspri offspring have the desired genotype βH S βH S αH αH NeoR NeoR NeoR NeoR βH S βH S αH αH NeoR NeoR NeoR NeoR 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 βH αH S βH αH S neo Neo Neneo R oR neo NeoR neo NeoR 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 βM βH αH S βH αH S neo Neo Neneo R oR neo NeoR neo Neo βM R βH αH S βH αH S neo Neo Neneo R oR neo NeoR neo NeoR x βM αM αM βH αH S βH αH S neo neo NeoR NeoR βM αM αM βH αH S βH αH S neo neo Neo NeoR R x βM αM αM βH αH S βH αH S neo neo NeoR NeoR βM αM αM βH αH S βH αH S neo neo NeoR NeoR βM αM αM βH αH S βH αH S neo neo Neo NeoR R βM αM αM βH αH S βH αH S neo neo Neo NeoR R There are many different mating schemes that one could use to generate mice that are homozygous for deletions in both the mouse α-globin gene and the
mouse B-globin gene,and that also carry the trangenes encoding the human a-globin gene and the human B-globin gene with the sickle cell mutation.What I have shown you is just one way to obtain this mouse.It should be noted that after birth,this mouse ONLY expressed human hemoglobin,and the mouse is therefore said to be humanized. Was it all worth it?Do we have a The outstanding news is that this mouse Sickle Cell Disease mouse model? does indeed represent an excellent model of Sickle Cell Disease which is now being used to explore therapies for SCD that are NeoR— very difficult to carry out on human Sickled Mouse RBCs SCD mouse has huge Mouse RBCs Sickle!! populations.So far,these mice have been clog the small blood spleen_workirg vessels in tissues overtime to clear defective RBCs used to explore the effectiveness of new drugs in ameliorating the tendency of RBCs Images removed due to copyright reasons. to sickle.Moreover,the mouse has been used to test out Gene Therapy approaches SCD 时 to treating the disease.Both of these approaches have been successful in the mouse,paving the way for trying out these treatments in people. Circulating RBCs ·Isolate mouse Bone Marrow stem cells Images removed due to copyright reasons. Images removed due to copyright reasons. ·Transfect with Please see figure 4 in lyamu,E.W.,E.A.Turner,and T.Asakura Human B-globin "Niprisan(Nix-0699)Improves the Survival Rates of Transgenic gene that produces Sickle Cell Mice Under Acute Severe Hypoxic Conditions.' a protein that Br J Haemato/.122,no.6(Sep.2003):1001-8. Kidney tissue damage prevents sickling ·Put the modified bone marrow back into a mouse ·Monitor Sickle Cell SCD Mouse expression of the Disease (SCD) After Gene transgene and the Lung of control mice Lung of mice taking Niprisan Mouse Therapy health of the mouse
mouse β-globin gene, and that also carry the trangenes encoding the human α-globin gene and the human β-globin gene with the sickle cell mutation. What I have shown you is just one way to obtain this mouse. It should be noted that after birth, this mouse ONLY expressed human hemoglobin, and the mouse is therefore said to be humanized. The outstanding news is that this mouse does indeed represent an excellent model of Sickle Cell Disease which is now being used to explore therapies for SCD that are very difficult to carry out on human populations. So far, these mice have been used to explore the effectiveness of new drugs in ameliorating the tendency of RBCs to sickle. Moreover, the mouse has been used to test out Gene Therapy approaches to treating the disease. Both of these approaches have been successful in the mouse, paving the way for trying out these treatments in people. βH S βH S αH αH NeoR NeoR NeoR NeoR Was it all worth it? Do we have a Sickle Cell Disease mouse model? Mouse RBCs Sickle!! Sickled Mouse RBCs clog the small blood vessels in tissues SCD wt SCD mouse has huge spleen…working overtime to clear defective RBCs βH S βH S αH αH NeoR NeoR NeoR NeoR βH S βH S αH αH NeoR NeoR NeoR NeoR Was it all worth it? Do we have a Sickle Cell Disease mouse model? Mouse RBCs Sickle!! Sickled Mouse RBCs clog the small blood vessels in tissues SCD wt SCD mouse has huge spleen…working overtime to clear defective RBCs Lung of control mice Lung of mice taking Niprisan Kidney tissue damage Circulating RBCs Sickle Cell Disease (SCD) Mouse SCD Mouse After Gene Therapy • Isolate mouse Bone Marrow stem cells • Transfect with Human β-globin gene that produces a protein that prevents sickling • Put the modified bone marrow back into a mouse • Monitor expression of the transgene and the health of the mouse Kidney tissue damage Circulating RBCs Kidney tissue damage Circulating RBCs Sickle Cell Disease (SCD) Mouse SCD Mouse After Gene Therapy • Isolate mouse Bone Marrow stem cells • Transfect with Human β-globin gene that produces a protein that prevents sickling • Put the modified bone marrow back into a mouse • Monitor expression of the transgene and the health of the mouse Images removed due to copyright reasons. Images removed due to copyright reasons. Images removed due to copyright reasons. Please see figure 4 in Iyamu, E. W., E. A. Turner, and T. Asakura. Br J Haematol. 122, no. 6 (Sep. 2003): 1001-8. "Niprisan (Nix-0699) Improves the Survival Rates of Transgenic Sickle Cell Mice Under Acute Severe Hypoxic Conditions