Table of Contents
Chapter 15. Transgenes in multicellular model organisms
In the next two chapters we will examine some of the ways in which we can study geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) function in higher eukaryotesplugin-autotooltip__default plugin-autotooltip_bigeukaryote: organism whose cells have membrane bound organelles, including the nucleus., using the laboratory mouse Mus musculus as an example. A remarkable number of manipulations have been made to the mouse genomeplugin-autotooltip__default plugin-autotooltip_bigGenome: a dataset that contains all DNA information of an organism. Most of the time, this also includes annotation and curation of that information, e.g., the names, locations, and functions of genes within the genome. As an adjective (“genomic”), this usually is used in the context of 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 transgenicplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. and knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. 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 missenseplugin-autotooltip__default plugin-autotooltip_bigMissense mutation: a mutation that causes a change in an the amino acid specified at a particular location in the gene sequence. mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. in a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that encodes one of the subunits of the proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. hemoglobin (Hb), namely β-globin. Hemoglobin is a tetrameric proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. made up of two α-globin polypeptides, and two β-globin polypeptides; the tetramer can be written as ααββ (Fig. 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.
The missenseplugin-autotooltip__default plugin-autotooltip_bigMissense mutation: a mutation that causes a change in an the amino acid specified at a particular location in the gene sequence. mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. that changes the seventh amino acidplugin-autotooltip__default plugin-autotooltip_bigAmino acid: molecules that are polymerized to form proteins. in β-globin from glutamine to valine causes devastating consequences. β-globin subunits that contain the sickle cell mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. are called βs, and Hb proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. that contain βs are called HbS (Fig. 1A). HbS does not directly interfere with the ability of hemoglobin to store or release oxygen, but rather this amino acidplugin-autotooltip__default plugin-autotooltip_bigAmino acid: molecules that are polymerized to form proteins. 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. 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. 1C). It is the ααβsβs hemoglobin molecule that is responsible for aggregating and causing sickle cell disease. The ααββs hemoglobin tetramers expressedplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. in people heterozygousplugin-autotooltip__default plugin-autotooltip_bigHeterozygous: a state for a diploid organism wherein the two alleles for a gene are different from each other. for the sickle mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. do not aggregate to form fibers, and so do not cause disease; however, if such heterozygousplugin-autotooltip__default plugin-autotooltip_bigHeterozygous: a state for a diploid organism wherein the two alleles for a gene are different from each other. people live at high altitude some sickling can occur (sometimes when an alleleplugin-autotooltip__default plugin-autotooltip_bigAllele: a version of a gene. Alleles of a gene are different if they have differences in their DNA sequence. is only weakly dominantplugin-autotooltip__default plugin-autotooltip_bigDominant: used to describe an allele, usually in comparison to wildtype. Dominant alleles will express their phenotype when combined with a wildtype allele. we can describe that as being semidominantplugin-autotooltip__default plugin-autotooltip_bigSemidominant: an allele $X$ is semidominant if a $\frac{X}{X}$ homozygous individual is fully mutant, a $\frac{X}{x}$ individual is partially or weakly mutant, and a $\frac{x}{x}$ individual is wildtype.).
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 alleleplugin-autotooltip__default plugin-autotooltip_bigAllele: a version of a gene. Alleles of a gene are different if they have differences in their DNA sequence. is actually quite surprising since one would expect it to be selected against as the human population expanded. However, people who are heterozygousplugin-autotooltip__default plugin-autotooltip_bigHeterozygous: a state for a diploid organism wherein the two alleles for a gene are different from each other. for the sickle mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. in the β-globin geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) are resistant to malaria. This gives a survival advantage for people who are carriers (i.e., heterozygotesplugin-autotooltip__default plugin-autotooltip_bigHeterozygous: a state for a diploid organism wherein the two alleles for a gene are different from each other.) of the mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. alleleplugin-autotooltip__default plugin-autotooltip_bigAllele: a version of a gene. Alleles of a gene are different if they have differences in their DNA sequence. 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.
Organization and expression of human globin genes
Mammals have several different β-globin-like genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), and a number of α-globin-like genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), i.e., a β-globin family and an α-globin family of genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-). These two geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) families are found on separate chromosomesplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins.; some of the family members are pseudogenesplugin-autotooltip__default plugin-autotooltip_bigPsuedogene: a DNA sequence that resembles a gene but cannot code for a protein, likely due to accumulated mutations over evolutionary time. (genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that do not produce functional proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes.), and the functional genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) in the family are expressedplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. at different times during development. For instance, the ααβsβs hemoglobin molecule that is responsible for aggregating and causing sickle cell disease is expressedplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. after birth.
How did all of these globin genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) appear in mammalian genomesplugin-autotooltip__default plugin-autotooltip_bigGenome: a dataset that contains all DNA information of an organism. Most of the time, this also includes annotation and curation of that information, e.g., the names, locations, and functions of genes within the genome. As an adjective (“genomic”), this usually is used in the context of, and what are they doing there? Many genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) in mammals exist as multi-geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) families, and the globin genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) are a good example of this. During mammalian evolution it appears that geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) duplication was a common event. This has allowed the duplicated genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) to accumulate mutationsplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. that sometimes inactivate the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (leading to pseudogenesplugin-autotooltip__default plugin-autotooltip_bigPsuedogene: a DNA sequence that resembles a gene but cannot code for a protein, likely due to accumulated mutations over evolutionary time.), but other times leads to genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that produce proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. that can carry out a slightly different function. Soon after duplication of an ancestral geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) to create the α-globin and β-globin ancestral genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), these two genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) were somehow moved to separate chromosomesplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. where they evolved their own geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) families through further duplication and mutationsplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. through evolutionary time.
Currently, the only known cure for sickle cell anemia is a bone marrow stem cellplugin-autotooltip__default plugin-autotooltip_bigStem cell: a cell that has the potential to adapt different cell fates to form different kinds of tissues. Not all stem cells are embryonic stem cells. transplant, which is technically difficult and expensive. While stem cellplugin-autotooltip__default plugin-autotooltip_bigStem cell: a cell that has the potential to adapt different cell fates to form different kinds of tissues. Not all stem cells are embryonic stem cells. treatments are promising, it's useful for us to consider historically how sickle cell anemia was studied to see how reverse geneticsplugin-autotooltip__default plugin-autotooltip_bigReverse genetics: an approach to studying genes wherein a researcher starts with knowledge of the physical identity of a gene (i.e., the DNA sequence of the gene) but does not know its function. In reverse genetics, the researcher uses various molecular genetic tools to create modified alleles that are reintroduced into an organism, with the goal of trying to deduce the function of the can be used to tackle problems in human disease. We will be discussing two general reverse geneticplugin-autotooltip__default plugin-autotooltip_bigReverse genetics: an approach to studying genes wherein a researcher starts with knowledge of the physical identity of a gene (i.e., the DNA sequence of the gene) but does not know its function. In reverse genetics, the researcher uses various molecular genetic tools to create modified alleles that are reintroduced into an organism, with the goal of trying to deduce the function of the approaches that can be applied to this problem - transgenicsplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. (this chapter) and knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. (covered in Chap. 16). The precise details of how we generate transgenicsplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. or knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. is not important here, although we will talk about them in a general sense - what we care about are the concepts of what transgenicsplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. and knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. can be used for in analyzing geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of 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 alleleplugin-autotooltip__default plugin-autotooltip_bigAllele: a version of a gene. Alleles of a gene are different if they have differences in their DNA sequence. 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 geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) with the sickle mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. (βSH, where the superscript H indicates human), in the hope that if the βSH proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. was expressedplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. at high levels it would precipitate Hb fibers that would cause sickling of RBCs, thus mimicking sickle cell disease.
How does one make a transgenicplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. 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 pronucleiplugin-autotooltip__default plugin-autotooltip_bigPronucleus: In a zygote, the nuclei of the sperm and oocyte that have not yet fused to form a diploid nucleus. (one from the mother and one from the father that have not yet fused to form the nucleusplugin-autotooltip__default plugin-autotooltip_bigNucleus: in eukaryotes, the membrane-bound organelle in cells that contains the chromosomes. of the zygoteplugin-autotooltip__default plugin-autotooltip_bigZygote: a cell formed by the fertilization of an oocyte by a sperm.) are still at the one-cell stage and are identified under the microscope. The male pronucleusplugin-autotooltip__default plugin-autotooltip_bigPronucleus: In a zygote, the nuclei of the sperm and oocyte that have not yet fused to form a diploid nucleus. is injected under the microscope with purified DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. fragments that contain the βSH geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) along with an appropriate upstreamplugin-autotooltip__default plugin-autotooltip_bigUpstream/downstream: These descriptors have different meanings depending on context:
* In genetics, these are terms used to describe directions on DNA, usually relative to the transcription start site of a gene. DNA sequences that are located in the same direction as the direction of regulatory region (promoterplugin-autotooltip__default plugin-autotooltip_bigPromoter: has multiple closely related but subtly different meanings depending on context:
* In bacteria, a promoter is a cis-acting DNA sequence near the transcription start site of a gene or operon that binds to bacterial RNA polymerase. * In eukaryotes, the formal definition of a promoter (also called a basal promoter) is a RNA plus enhancersplugin-autotooltip__default plugin-autotooltip_bigEnhancer: a more inclusive term for a UAS. One reason this is a more inclusive term is because not all enhancers are located upstream of genes; some enhancers are located downstream of a gene, and in some cases can even be located inside a gene.) to give it a good chance of being expressedplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places.. The injected DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. quite often gets integrated into the genomeplugin-autotooltip__default plugin-autotooltip_bigGenome: a dataset that contains all DNA information of an organism. Most of the time, this also includes annotation and curation of that information, e.g., the names, locations, and functions of genes within the genome. As an adjective (“genomic”), this usually is used in the context of, and about one in three eggs that are implanted into a foster mother mouse will have the βSHv geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) integrated. These eggs will go on to produce baby mice, and we can confirm that the transgeneplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. has been integrated into the genomeplugin-autotooltip__default plugin-autotooltip_bigGenome: a dataset that contains all DNA information of an organism. Most of the time, this also includes annotation and curation of that information, e.g., the names, locations, and functions of genes within the genome. As an adjective (“genomic”), this usually is used in the context of by using techniques such as PCRplugin-autotooltip__default plugin-autotooltip_bigPolymerase chain reaction (PCR): An experimental technique invented by Kary Mullis used to exponentially amplify DNA in vitro. PCR made obtaining large quantities of DNA for analysis much faster and easier than using traditional cloning methods. (Chap. 07). Usually, these foundersplugin-autotooltip__default plugin-autotooltip_bigFounder: when making genetically modified organisms, a founder is an individual from the first generation of organisms that are expected to carry the desired genetic modification in the germline, such that the genetic modification has the potential to be passed down to offspring. are then bred to ensure that the transgeneplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. has been incorporated into the germlineplugin-autotooltip__default plugin-autotooltip_bigGermline: cells and tissue of the reproductive system. so that a transgenicplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. lineplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and can be established and propagated for future use. Transgenicplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. mice from such a lineplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and express the mutated human β-globin proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. in their RBCs (this is usually confirmed through other experiments).
Scientists found that these transgenicplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. mice were not a good model for sickle cell disease. It turns out that the human β-globin proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. does not complex well with the mouse α-globin proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. (αM; superscript M stands for mouse), probably due to differences between humans and mice. To try and get around this problem, the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) encoding the human α-globin proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. (αH) was used to create a new transgenicplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. mouse lineplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and, which was then mated with the βSH transgenicplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. mouse to produce a mouse expressing both βSH and αH human proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes.. The expectation of this experiment was that the presence of the αHαHβSHβSH 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 proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. prevented the mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. hemoglobin tetramers from precipitating into fibers; these transgenicplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. 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 genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) by geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics., discussed in the next chapter.
Questions and exercises
Conceptual question: We discussed transgenesplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. (by using plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors.) and geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. in yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 in Chapter 14. What are some of the technical challenges in making transgenicplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. mice compared to transgenicplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02?
Conceptual question: How can you determine if your transgeneplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. integrates into some other important geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-)? In other words, your transgeneplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. might have some effect on the mouse, but how do you know that it's due to expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of the transgeneplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. itself and not caused by disrupting another geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) located where it integrates?
Exercise 1: Assume you have generated the two transgenicplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. mouse lines described above: αH and β<sub>SH. How will you breed the two lines to generate the double transgenicplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. lineplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and? Assume you want to make the two transgenesplugin-autotooltip__default plugin-autotooltip_bigTransgene: a gene usually from one organism that is introduced into a different organism for research purposes. Although technically plasmids and other episomes can carry transgenes into E. coli, yeast, or other microbes, the term “transgene” tends to be used more for multicellular organisms. double homozygousplugin-autotooltip__default plugin-autotooltip_bigHomozygous: a state for a diploid organism wherein the two alleles for a gene are identical to each other..