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--- chapter_15 [2024/08/23 18:56] – mike
+++ chapter_15 [2025/05/04 19:53] (current) – [Human sickle cell disease: an introduction] mike
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-In the next two chapters we will examine some of the ways in which we can study gene function in higher eukaryotes, more specifically in the laboratory mouse Mus musculus. 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.
+<-chapter_14|Chapter 14^table_of_contents|Table of Contents^chapter_16|Chapter 16->
-===== Human sickle cell disease: an introduction =====
+<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 (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 -globin. Hemoglobin is a tetrameric protein made up of two -globin polypeptides, and two -globin polypeptides; the tetramer can be written as (Fig. 15.1A). 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: an introduction =====
-Figure 15.1. Sickle cell disease. (A) Hemoglobin is a tetramer comprised of two  subunits and two  subunits. In sickle cell disease, a mutation alters the  subunit. (B) Red blood cells (RBCs) become sickled when oxygen levels are low. (C) Sickled RBCs can block small blood vessels, leading to tissue damage, such as in kidneys. Source: Genes and Disease. Licensing: Public domain.
-The missense mutation that changes the seventh amino acid in β-globin from glutamine to valine causes devastating consequences. -globin subunits that contain the sickle cell mutation are called s, and Hb proteins that contain s are called HbS (Fig. 15.1A). 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. 15.1B). 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. 15.1C). It is the ss hemoglobin molecule that is responsible for aggregating and causing sickle cell disease. The s 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).
+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.
-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 -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.
+<figure Fig1>
+{{ :sickle.jpg?400 |}}
+<caption>
+Sickle cell disease. (A) Hemoglobin is a tetramer comprised of two α subunits and two β subunits. In sickle cell disease, a mutation alters the β subunit. (B) Red blood cells (RBCs) become sickled when oxygen levels are low. (C) Sickled RBCs can block small blood vessels, leading to tissue damage, such as in kidneys. Source: [[https://www.ncbi.nlm.nih.gov/books/NBK22183/|Genes and Disease]], National Center for Biotechnology Information. Licensing: Public domain.
+</caption>
+</figure>
+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.
-Figure 15.2. Coincidence of HbS allele frequency and malaria prevalence. Source: Piel et al. (2010) Nat. Comm. 1:104. DOI: 10.1038/ncomms1104. Licensing: CC BY-NC-SA 3.0.
+<figure Fig2>
+{{ :hbs_allele_distribution.jpg?400 |}}
+<caption>
+Coincidence of HbS allele frequency and malaria prevalence. Source: Piel et al. (2010) Nat. Comm. 1:104. http://doi.org/10.1038/ncomms1104. Licensing: [[https://creativecommons.org/licenses/by-nc-sa/3.0/|CC BY-NC-SA 3.0]].
+</caption>
+</figure>
 ===== Organization and expression of human globin genes =====
-Mammals have several different -globin-like genes, and a number of -globin-like genes, i.e., a -globin family and an -globin family of genes. These two gene families are found on separate chromosomes; some of the family members are pseudogenes (genes that do not produce functional proteins), and the functional genes in the family are expressed at different times during development. For instance, the ss hemoglobin molecule that is responsible for aggregating and causing sickle cell disease is expressed after birth.
+Mammals have several different β-globin-like genes, and a number of α-globin-like genes, i.e., a β-globin family and an α-globin family of genes. These two gene families are found on separate chromosomes; some of the family members are pseudogenes (genes that do not produce functional proteins), and the functional genes in the family are expressed at different times during development. For instance, the ααβ<sub>s</sub>β<sub>s</sub> hemoglobin molecule that is responsible for aggregating and causing sickle cell disease is expressed after birth.
-How did all of these globin genes appear in mammalian genomes, and what are they doing there? Many genes in mammals exist as multi-gene families, and the globin genes are a good example of this. During mammalian evolution it appears that gene duplication was a common event. This has allowed the duplicated genes to accumulate mutations that sometimes inactivate the gene (leading to pseudogenes), but other times leads to genes that produce proteins that can carry out a slightly different function. Soon after duplication of an ancestral gene to create the -globin and -globin ancestral genes, these two genes were somehow moved to separate chromosomes where they evolved their own gene families through further duplication and mutations through evolutionary time.
+How did all of these globin genes appear in mammalian genomes, and what are they doing there? Many genes in mammals exist as multi-gene families, and the globin genes are a good example of this. During mammalian evolution it appears that gene duplication was a common event. This has allowed the duplicated genes to accumulate mutations that sometimes inactivate the gene (leading to pseudogenes), but other times leads to genes that produce proteins that can carry out a slightly different function. Soon after duplication of an ancestral gene to create the α-globin and β-globin ancestral genes, these two genes were somehow moved to separate chromosomes where they evolved their own gene families through further duplication and mutations through evolutionary time.
+<figure Fig3>
-Figure 15.3. The origin of the globin gene family in mammals.
+{{ :globin_gene_family.png?400 |}}
-Currently, the only known cure for sickle cell anemia is a bone marrow stem cell transplant, which is technically difficult and expensive. While stem cell treatments are promising, it's useful for us to consider historically how sickle cell anemia was studied to see how reverse genetics can be used to tackle problems in human disease. We will be discussing two general reverse genetic approaches that can be applied to this problem - transgenics (this chapter) and knockouts (covered in Chap. 16). The precise details of how we generate transgenics or knockouts is not important here, although we will talk about them in a general sense - what we care about are the concepts of what transgenics and knockouts can be used for in analyzing gene function.
+<caption>
+The origin of the globin gene family in mammals.
+</caption>
+</figure>
-===== Making transgenic mice that express mutant human -globin =====
+Currently, the only known cure for sickle cell anemia is a bone marrow stem cell transplant, which is technically difficult and expensive. While stem cell treatments are promising, it's useful for us to consider historically how sickle cell anemia was studied to see how reverse genetics can be used to tackle problems in human disease. We will be discussing two general reverse genetic approaches that can be applied to this problem - transgenics (this chapter) and knockouts (covered in [[chapter_16|Chap. 16]]). The precise details of how we generate transgenics or knockouts is not important here, although we will talk about them in a general sense - what we care about are the concepts of what transgenics and knockouts can be used for in analyzing gene function.
+===== Making transgenic mice that express mutant human β-globin =====
-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 (βSH, where the superscript H indicates human), in the hope that if the SH 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 SH 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 SH 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 (Chap. 7.5). 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).
+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. 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).
-Figure 15.4. Strategy for generating transgenic mice.
-Scientists found that these transgenic mice were not a good model for sickle cell disease. It turns out that the human -globin protein does not complex well with the mouse - globin protein (M; superscript M stands for mouse), probably due to differences between humans and mice. To try and get around this problem, the gene encoding the human -globin protein (H) was used to create a new transgenic mouse line, which was then mated with the SH transgenic mouse to produce a mouse expressing both SH and H human proteins. The expectation of this experiment was that the presence of the HHSHSH hemoglobin tetramer in mouse RBCs would lead to the precipitation of fibers and the sickling of the mouse RBCs.
-Discussion Box: How can you determine if your transgene integrates into some other important gene? In other words, your transgene might have some effect on the mouse, but how do you know that it's due to expression of the transgene itself and not caused by disrupting another gene located where it integrates?
+<figure>
+{{ :mouse_transgenesis.png?400 |}}
+<caption>
+Strategy for generating transgenic mice.
+</caption>
+</figure>
+Scientists found that these transgenic mice were not a good model for sickle cell disease. It turns out that the human β-globin protein does not complex well with the mouse α-globin protein (α<sup>M</sup>; superscript M stands for mouse), probably due to differences between humans and mice. To try and get around this problem, the gene encoding the human α-globin protein (α<sup>H</sup>) was used to create a new transgenic mouse line, which was then mated with the β<sub>S</sub><sup>H</sup> transgenic mouse to produce a mouse expressing both β<sub>S</sub><sup>H</sup> and α<sup>H</sup> human proteins. The expectation of this experiment was that the presence of the α<sup>H</sup>α<sup>H</sup>β<sub>S</sub><sup>H</sup>β<sub>S</sub><sup>H</sup> hemoglobin tetramer in mouse RBCs would lead to the precipitation of fibers and the sickling of the mouse RBCs.
 However, much to the disappointment of the scientists, this was not the case. They found that the normal mouse hemoglobin proteins prevented the mutant hemoglobin tetramers from precipitating into fibers; these transgenic mice were not a good model for human sickle cell disease. The scientists decided that the only solution to this problem would be to eliminate the endogenous mouse α and β globin genes by gene knockouts, discussed in the next chapter.
@@ Line 45: / Line 62: @@
-Discussion box: We discussed transgenes (by using plasmids) and gene knockouts in yeast in Chapter 14. What are some of the technical challenges in making transgenic mice compared to transgenic yeast?
+Conceptual question: We discussed transgenes (by using plasmids) and gene knockouts in yeast in [[chapter_14|Chapter 14]]. What are some of the technical challenges in making transgenic mice compared to transgenic yeast?
+Conceptual question: How can you determine if your transgene integrates into some other important gene? In other words, your transgene might have some effect on the mouse, but how do you know that it's due to expression of the transgene itself and not caused by disrupting another gene located where it integrates?
+Exercise 1: Assume you have generated the two transgenic mouse lines described above: α<sup>H</sup> and β<sub><sub>S</sub><sup>H</sup>. How will you breed the two lines to generate the double transgenic line? Assume you want to make the two transgenes double homozygous.