Unformatted text preview: 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 -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. 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.
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the last lecture
Inject foreign DNA into one of the pronuclei Pronuclei M
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M M 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 S-globin gene recombine with the mouse -globin gene? Could one inject the w.t. human -globin gene into a human embryo to correct the deficiency?
Figure by MIT OCW. M Fertilized mouse egg prior to fusion of male and female pronuclei Transfer injected eggs into foster mother. PROBLEM: These mice still do not have RBCs that sickle very well. The mouse still has mouse and 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 and globin genes by targeted homologous recombination to generate "Knock-out" mice 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 So...how do we "get rid of" the endogenous mouse -globin and -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 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 Early findings totipotent cells mix together and revealed that the produce an animal that has a preimplantation micture two types of cells in its mouse embryo body. This animal has four genetic Images removed due to is remarkably parents!! copyright reasons. 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 gene knock-out technology. 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 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!!
neor tkHSV 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 Construct
Nonhomologous recombination Gene X replacement construct ES cells ES cells Homologous recombination ES-cell DNA Other genes Random insertion Gene X ES-cell DNA Gene-targeted insertion No mutation in gene X Mutation in gene X Cells are resistant to G-418 and ganciclovir Can remove totipotent EMBRYONIC STEM CELLS (ES cells) and culture in vitro Select for the genetically altered cells you want Cells are resistant to G-418 but sensitive to ganciclovir Formation of ES Cells Carrying a Knockout Mutation Targeting Construct
R TK V HS 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 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 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. 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 H eterozygous for the knocked out gene H eterozygous for the knocked out gene M +/- M +/- 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. M +/+
25% M +/50% M -/25%
H om ozygous m utant m ice... 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 -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 -globin gene and then again for deletion mutations in the -globin gene.
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H S SPERM ++ +- -+ -- ++ H
M M M +/+ +/+ +/+ +/- SOLUTION: Need to get rid of the endogenous mouse and globin genes by targeted homologous recombination to generate "Knock-out" mice +/- +/+ +/- +/- EGGS neo M NeoR
neo NeoR H S Neo
H S neoR H
H +- +/+ +/- +/+ -/- +/- +/- +/- -/- is essentially an This is essentially an X AaBb cross AaBb X AaBb cross the A and B genes where the A and B genes on different lie on different chromosomes and are chromosomes and are therefore unlinked. therefore unlinked. Human transgenes The Human transgenes homozygous in both are homozygous in both parents and so will be parents and so will be present in all offspring. present in all offspring.
1/16 offspring have the 1/16 offspring have the desired genotype desired genotype Neo neoR -+ +/- +/+ +/- +/- -/- +/+ -/- +/- H S H
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H S -- +/- +/- +/- -/- -/- +/- -/- -/- Neo Neo neo R H
H H S S H H
NeoR Neo R x
H S H
M M Neo R
Neo R M
NeoR H H S S H H neo H S NeoR neo H NeoR Neo R There are many different mating schemes that one could use to generate mice that are homozygous for deletions in both the mouse -globin gene and the mouse -globin gene, and that also carry the trangenes encoding the human -globin gene and the human -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 H Sickle Cell Disease which is now being Neo Neo used to explore therapies for SCD that are H very difficult to carry out on human Neo Neo Sickled Mouse RBCs SCD mouse has huge populations. So far, these mice have been Mouse RBCs Sickle!! clog the small blood spleen...working vessels in tissues overtime to clear used to explore the effectiveness of new defective RBCs drugs in ameliorating the tendency of RBCs to sickle. Moreover, the mouse has been Images removed due to copyright reasons. used to test out Gene Therapy approaches to treating the disease. Both of these SCD wt approaches have been successful in the mouse, paving the way for trying out these treatments in people.
Was it all worth it? Do we have a Sickle Cell Disease mouse model?
H S H S R R R R Circulating RBCs Isolate mouse Bone Marrow stem cells Human -globin gene that produces a protein that prevents sickling Put the modified bone marrow back into a mouse Images removed due to copyright reasons. Please see figure 4 in Iyamu, E. W., E. A. Turner, and T. Asakura. "Niprisan (Nix-0699) Improves the Survival Rates of Transgenic Sickle Cell Mice Under Acute Severe Hypoxic Conditions." Br J Haematol. 122, no. 6 (Sep. 2003): 1001-8. Images removed due to copyright reasons. Transfect with Kidney tissue damage Lung of control mice Lung of mice taking Niprisan Sickle Cell Disease (SCD) Mouse SCD Mouse After Gene Therapy Monitor expression of the transgene and the health of the mouse ...
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