Differences

This shows you the differences between two versions of the page.

--- chapter_16 [2025/05/15 13:30] – [Creating a mouse model] mike
+++ chapter_16 [2025/05/16 21:53] (current) – [Creating knockout mice] mike
@@ Line 6: / Line 6: @@
-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>
@@ Line 29: / Line 29: @@
 </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 =====
@@ Line 62: / Line 62: @@
 ===== 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>