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--- chapter_15 [2024/08/23 21:41] – [Questions and exercises] mike
+++ chapter_15 [2025/05/04 19:53] (current) – [Human sickle cell disease: an introduction] mike
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-<typo fs:x-large>Chapter 15. Transgenes in multicellular model organisms</typo>
+<-chapter_14|Chapter 14^table_of_contents|Table of Contents^chapter_16|Chapter 16->
-In the next two chapters we will examine some of the ways in which we can study gene function in higher eukaryotes, using the laboratory mouse //Mus musculus// as an example. A remarkable number of manipulations have been made to the mouse genome in order to generate an experimental mouse model system for human sickle cell disease. The mouse that was developed to explore this human disease turns out to be one of most genetically modified mice on the planet! It gives us an interesting framework in which to discuss making transgenic and knockout mice. To set the scene for genetically modifying mice to mimic human sickle cell disease we need to step back a bit and consider this devastating human disease and some of its features.
+<typo fs:x-large>Chapter 15. %%Transgenes%% in multicellular model organisms</typo>
+In the next two chapters we will examine some of the ways in which we can study gene function in higher eukaryotes, using the laboratory mouse //Mus musculus// as an example. A remarkable number of manipulations have been made to the mouse genome in order to generate an experimental mouse model system for human sickle cell disease. The mouse that was developed to explore this human disease turns out to be one of the most genetically modified mice on the planet! It gives us an interesting framework in which to discuss making transgenic and knockout mice. To set the scene for genetically modifying mice to mimic human sickle cell disease we need to step back a bit and consider this devastating human disease and some of its features.
 ===== Human sickle cell disease: an introduction =====
-Human sickle cell disease (also called sickle cell anemia) is a human blood disorder that is caused by a single missense mutation in a gene that encodes one of the subunits of the protein hemoglobin (Hb), namely β-globin. Hemoglobin is a tetrameric protein made up of two α-globin polypeptides, and two α-globin polypeptides; the tetramer can be written as ααββ (Fig. {{ref>Fig1}}A). Each of the four globin polypeptides bind to an iron-containing heme molecule (iron is what makes hemoglobin and red blood cells red) whose function is to bind oxygen in the lungs and release it in all the tissues of the animal.
+Human sickle cell disease (also called sickle cell anemia) is a human blood disorder that is caused by a single missense mutation in a gene that encodes one of the subunits of the protein hemoglobin (Hb), namely β-globin. Hemoglobin is a tetrameric protein made up of two α-globin polypeptides, and two β-globin polypeptides; the tetramer can be written as ααββ (Fig. {{ref>Fig1}}A). Each of the four globin polypeptides bind to an iron-containing heme molecule (iron is what makes hemoglobin and red blood cells red) whose function is to bind oxygen in the lungs and release it in all the tissues of the animal.
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-The missense mutation that changes the seventh amino acid in β-globin from glutamine to valine causes devastating consequences. β-globin subunits that contain the sickle cell mutation are called β<sub>s</sub>, and Hb proteins that contain β<sub>s</sub> are called HbS (Fig. {{ref>Fig1}}A). HbS does not directly interfere with the ability of hemoglobin to store or release oxygen, but rather this amino acid change bestows a new property on the hemoglobin molecule; in its deoxygenated state the HbS molecules aggregate together to form polymeric fibers, and the presence of these fibers grossly distorts the shape of red blood cells (RBCs) (Fig. {{ref>1}}B). Instead of the classic dual-concave round shape that has tremendous flexibility to squeeze through tiny capillaries within tissues, the aggregated HbS fibers cause the RBCs to become curved (like a sickle), rigid, prone to rupture, and prone to clumping; rupture causes anemia (a lack of healthy red blood cells to carry oxygen to tissues) and clumping clogs small blood vessels, leading to tissue damage (Fig. {{ref>Fig1}}1C). It is the ααβ<sub>s</sub>β<sub>s</sub> hemoglobin molecule that is responsible for aggregating and causing sickle cell disease. The ααββ<sub>s</sub> hemoglobin tetramers expressed in people heterozygous for the sickle mutation do not aggregate to form fibers, and so do not cause disease; however, if such heterozygous people live at high altitude some sickling can occur (sometimes when an allele is only weakly dominant we can describe that as being semidominant).
+The missense mutation that changes the seventh amino acid in β-globin from glutamine to valine causes devastating consequences. β-globin subunits that contain the sickle cell mutation are called β<sub>s</sub>, and Hb proteins that contain β<sub>s</sub> are called HbS (Fig. {{ref>Fig1}}A). HbS does not directly interfere with the ability of hemoglobin to store or release oxygen, but rather this amino acid change bestows a new property on the hemoglobin molecule; in its deoxygenated state the HbS molecules aggregate together to form polymeric fibers, and the presence of these fibers grossly distorts the shape of red blood cells (RBCs) (Fig. {{ref>Fig1}}B). Instead of the classic dual-concave round shape that has tremendous flexibility to squeeze through tiny capillaries within tissues, the aggregated HbS fibers cause the RBCs to become curved (like a sickle), rigid, prone to rupture, and prone to clumping; rupture causes anemia (a lack of healthy red blood cells to carry oxygen to tissues) and clumping clogs small blood vessels, leading to tissue damage (Fig. {{ref>Fig1}}C). It is the ααβ<sub>s</sub>β<sub>s</sub> hemoglobin molecule that is responsible for aggregating and causing sickle cell disease. The ααββ<sub>s</sub> hemoglobin tetramers expressed in people heterozygous for the sickle mutation do not aggregate to form fibers, and so do not cause disease; however, if such heterozygous people live at high altitude some sickling can occur (sometimes when an allele is only weakly dominant we can describe that as being semidominant).
 Sickle cell disease is very common in many parts of the world, especially sub-Saharan Africa, and even among African Americans (1 in 365 births) and Hispanic Americans (1 in 16,300 births). The prevalence of such a devastating disease allele is actually quite surprising since one would expect it to be selected against as the human population expanded. However, people who are heterozygous for the sickle mutation in the β-globin gene are resistant to malaria. This gives a survival advantage for people who are carriers (i.e., heterozygotes) of the mutant allele if they live in an area where malaria is prevalent. These individuals are said to have the sickle cell trait, but they do not have sickle cell disease.
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-One way to study sickle cell disease is to generate an animal model of the disease such that it simulates the human disease. The animal model gives you a platform that you can use to test therapies or study the biology in more detail than you could with human patients. One way to generate a mouse model is through transgenesis - that is, we express the disease allele in a mouse in the hope that the mouse will have symptoms that resemble sickle cell disease. In the 1980s and early 1990s researchers tried to make a mouse with sickle cell disease by introducing the human -globin gene with the sickle mutation (β<sub>S</sub><sup>H</sup>, where the superscript H indicates human), in the hope that if the β<sub>S</sub><sup>H</sup> protein was expressed at high levels it would precipitate Hb fibers that would cause sickling of RBCs, thus mimicking sickle cell disease.
+One way to study sickle cell disease is to generate an animal model of the disease such that it simulates the human disease. The animal model gives you a platform that you can use to test therapies or study the biology in more detail than you could with human patients. One way to generate a mouse model is through transgenesis - that is, we express the disease allele in a mouse in the hope that the mouse will have symptoms that resemble sickle cell disease. In the 1980s and early 1990s researchers tried to make a mouse with sickle cell disease by introducing the human β-globin gene with the sickle mutation (β<sub>S</sub><sup>H</sup>, where the superscript H indicates human), in the hope that if the β<sub>S</sub><sup>H</sup> protein was expressed at high levels it would precipitate Hb fibers that would cause sickling of RBCs, thus mimicking sickle cell disease.
-How does one make a transgenic mouse? Female mice are treated with a hormone to make them super-ovulate and then are mated to males. Soon after mating, the fertilized eggs are surgically retrieved from the uterus. Eggs that contain two pronuclei (one from the mother and one from the father that have not yet fused to form the nucleus of the zygote) are still at the one-cell stage and are identified under the microscope. The male pronucleus is injected under the microscope with purified DNA fragments that contain the β<sub>S</sub><sup>H</sup> gene along with an appropriate upstream regulatory region (promoter plus enhancers) to give it a good chance of being expressed. The injected DNA quite often gets integrated into the genome, and about one in three eggs that are implanted into a foster mother mouse will have the β<sub>S</sub><sup>H</sup>v gene integrated. These eggs will go on to produce baby mice, and we can confirm that the transgene has been integrated into the genome by using techniques such as PCR ([[chapter_07|Chap. 7]]). Usually, these founders are then bred to ensure that the transgene has been incorporated into the germline so that a transgenic line can be established and propagated for future use. Transgenic mice from such a line express the mutated human β-globin protein in their RBCs (this is usually confirmed through other experiments).
+How does one make a transgenic mouse? Female mice are treated with a hormone to make them super-ovulate and then are mated to males. Soon after mating, the fertilized eggs are surgically retrieved from the uterus. Eggs that contain two pronuclei (one from the mother and one from the father that have not yet fused to form the nucleus of the zygote) are still at the one-cell stage and are identified under the microscope. The male pronucleus is injected under the microscope with purified DNA fragments that contain the β<sub>S</sub><sup>H</sup> gene along with an appropriate upstream regulatory region (promoter plus enhancers) to give it a good chance of being expressed. The injected DNA quite often gets integrated into the genome, and about one in three eggs that are implanted into a foster mother mouse will have the β<sub>S</sub><sup>H</sup>v gene integrated. These eggs will go on to produce baby mice, and we can confirm that the transgene has been integrated into the genome by using techniques such as PCR ([[chapter_07|Chap. 07]]). Usually, these founders are then bred to ensure that the transgene has been incorporated into the germline so that a transgenic line can be established and propagated for future use. Transgenic mice from such a line express the mutated human β-globin protein in their RBCs (this is usually confirmed through other experiments).
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