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--- chapter_16 [2024/09/01 23:37] – mike
+++ chapter_16 [2025/05/16 21:53] (current) – [Creating knockout mice] mike
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-Knocking out mouse genes, or deliberately targeting a specific gene for mutation (as opposed to creating random mutants with a chemical mutagen), is a much more complex process than making transgenic mice. To discuss this, some background information about the preimplantation mouse embryo is first needed. For about 4-5 days after fertilization, mouse embryos are free-floating in the uterus (which means we can surgically remove them) and many of the cells that will eventually form the mouse remain totipotent, meaning that they have the potential to differentiate into any kind of mouse cell type (Fig. 16.1). 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 (the cells will naturally stick together) 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 mixture two types of cells in its body. The ability of these genetically different totipotent cells to mix together in the preimplantation embryo is crucial for the mouse gene knockout technology.
+Knocking out mouse genes, or deliberately targeting a specific gene for mutation (as opposed to creating random mutants with a chemical mutagen), is a much more complex process than making transgenic mice. To discuss this, some background information about the preimplantation mouse embryo is first needed. For about 4-5 days after fertilization, mouse embryos are free-floating in the uterus (which means we can surgically remove them) and many of the cells that will eventually form the mouse remain totipotent, meaning that they have the potential to differentiate into any kind of mouse cell type (Fig. 16.1). 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 (the cells will naturally stick together) 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 mixture of two types of cells in its body. The ability of these genetically different totipotent cells to mix together in the preimplantation embryo is crucial for the mouse gene knockout technology.
 <figure Fig1>
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 In order to make a directed genetic change in a specific mouse gene we exploit homologous recombination. However, this is much harder to do in mammalian cells than bacteria and yeast. In yeast, when a linear fragment of dsDNA is introduced into a yeast cell, that DNA is integrated into the yeast genome by homologous recombination about 90% of the time, such that the incoming DNA fragment replaces the endogenous gene on the chromosome. In mammalian cells, DNA that is introduced into a cell almost always integrates at a non-homologous site (see [[chapter_15|Chap. 15]]), and the frequency of homologous recombination at the endogenous locus is very low - about 10<sup>-3</sup> to 10<sup>-5</sup> (or 0.1% - 0.001%). To have a chance at finding rare events like this, we need to have an experimental setup where we can:
   - look through thousands of independent integration events; and
-  - a way to be able to identify the specific integration event we want, namely an integration even that took place by homologous recombination.
+  - a way to be able to identify the specific integration event we want, namely an integration event that took place by homologous recombination.
 The first crucial technological advancement for creating mouse knockouts was being able to grow the totipotent cells from preimplantation embryos in culture in the lab; these cells are called mouse embryonic stem cells (ES cells). Cells from the inner cell mass of a preimplantation embryo at the blastocyst stage can be removed and cultured in a dish without the cells losing their totipotency (Fig. {{ref>Fig2}}); even after being cultured in the lab for many years these cells could still be introduced back into a preimplantation embryo and go on to make all the tissues of a mouse.
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 </figure>
-This gave scientists an opportunity to genetically manipulate ES cells in a Petri dish, in an analogous way as we might genetically manipulate yeast or //E. coli// cells in a Petri dish. Importantly, this further gives scientists the opportunity to isolate clones as colonies and also the ability to grow a large number of cells to screen for rare events such as homologous recombination (it is far easier and cheaper to grow thousands of cells in a dish than it is to grow thousands of mice in cages). Once you have identified mouse ES cells (originally from a gray furred mouse) that have been genetically altered the way you wish, these cells can be used to generate a living animal that is fully derived from these totipotent ES cells.
+This gave scientists an opportunity to genetically manipulate ES cells in a Petri dish, in an analogous way as we might genetically manipulate yeast or //E. coli// cells in a Petri dish. Importantly, this further gives scientists the opportunity to isolate clones as colonies and also the ability to grow a large number of cells to screen for rare events such as homologous recombination (it is far easier and cheaper to grow thousands of cells in a dish than it is to grow thousands of mice in cages). Once you have identified mouse ES cells (originally from a gray-furred mouse) that have been genetically altered the way you wish, these cells can be used to generate a living animal that is fully derived from these totipotent ES cells.
 ===== Selecting for homologous recombination in ES cells =====
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 ===== Creating knockout mice =====
-The next step is to create a mouse in which every cell in the mouse contains the genetic alteration you just created in ES cells. The first thing to note is that the ES cells in which we knocked out gene X are from a mouse strain that has gray fur. We will inject these ES cells into a new blastocyst embryo that comes from a mouse strain that has white fur (Fig. {{ref>Fig4}}). Usually, we will do this for a few dozen embryos, which are then implanted into foster mothers. Some pups born from these foster mothers will have both white and gray fur - they are chimeras. We then take male chimeric pups and breed them to white females. Since the gray fur phenotype is dominant, any chimeric father that has germ cells that formed from our modified ES cells will produce pups will all gray fur; these pups are then founders for a knockout mouse line (Fig. {{ref>Fig5}}).
+The next step is to create a mouse in which every cell in the mouse contains the genetic alteration you just created in ES cells. The first thing to note is that the ES cells in which we knocked out gene X are from a mouse strain that has gray fur. We will inject these ES cells into a new blastocyst embryo that comes from a mouse strain that has white fur (Fig. {{ref>Fig4}}). Usually, we will do this for a few dozen embryos, which are then implanted into foster mothers. Some pups born from these foster mothers will have both white and gray fur - they are chimeras. We then take male chimeric pups and breed them to white females. Since the gray fur phenotype is dominant, any chimeric father that has germ cells that formed from our modified ES cells will produce pups with all gray fur; these pups are then founders for a knockout mouse line (Fig. {{ref>Fig5}}).
 <figure Fig5>
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 Some final notes on our story: this humanized mouse did indeed represent an excellent model of sickle cell disease, which was used to explore therapies that are very difficult to carry out on humans. For instance, these mice were used to explore the effectiveness of new drugs in reducing the tendency of RBCs to sickle. Moreover, these mice have been used to test out gene therapy approaches to treating the disease. Although stem cell therapies have rendered some of these treatments obsolete, we use this story to illustrate the power of reverse genetics as a general strategy to study gene function or genetic diseases.
-We also note that while this chapter discusses the technique for how to make gene knockouts in mice, the idea of “reverse genetics”, i.e., knocking out a specific gene of interest (as opposed to randomly mutagenizing genes and looking for interesting mutants) is a general strategy for studying gene function. Within the technical limits of different experimental organisms, this general strategy can be applied to just about any commonly studied model genetic organism (such as //E. coli//, yeast, or Drosophila), although the precise technical details of how knockouts can be generated for each organism will of course be somewhat different. A new tool developed around 2012 called CRISPR (briefly discussed in [[chapter_17|Chapter 17]] has made this gene knockout approach for studying gene function much more accessible to organisms other than the traditional model organisms.
+We also note that while this chapter discusses the technique for how to make gene knockouts in mice, the idea of “reverse genetics”, i.e., knocking out a specific gene of interest (as opposed to randomly mutagenizing genes and looking for interesting mutants) is a general strategy for studying gene function. Within the technical limits of different experimental organisms, this general strategy can be applied to just about any commonly studied model genetic organism (such as //E. coli//, yeast, or Drosophila), although the precise technical details of how knockouts can be generated for each organism will of course be somewhat different. A new tool developed around 2012 called CRISPR (briefly discussed in [[chapter_17|Chapter 17]]) has made this gene knockout approach for studying gene function much more accessible to organisms other than the traditional model organisms.
 ===== Questions and exercises =====