Knocking out mouse genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), or deliberately targeting a specific geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) for 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. (as opposed to creating random mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. with a chemical mutagenplugin-autotooltip__default plugin-autotooltip_bigMutagen: a chemical or source of ionizing radiation that has the potential to damage DNA such that the DNA sequence will be altered.), is a much more complex process than 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. To discuss this, some background information about the preimplantationplugin-autotooltip__default plugin-autotooltip_bigPreimplantation: in mammals, a preimplantation embryo is an embryo that has not yet attached to the uterus. 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 totipotentplugin-autotooltip__default plugin-autotooltip_bigTotipotent: used to describe stem cells that can potentially differentiate into all cell types of an organism., 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 chimericplugin-autotooltip__default plugin-autotooltip_bigChimera: In the context of multicellular animals, a chimera is an individual that contains cells derived from multiple parents. In a general sense, “chimera” means something with a mixed constitution. In Greek mythology, a chimera is a monster that has a lion's head, a goat's body, and a serpent's tail. See also mouse will be born. Essentially the two types of totipotentplugin-autotooltip__default plugin-autotooltip_bigTotipotent: used to describe stem cells that can potentially differentiate into all cell types of an organism. 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 totipotentplugin-autotooltip__default plugin-autotooltip_bigTotipotent: used to describe stem cells that can potentially differentiate into all cell types of an organism. cells to mix together in the preimplantationplugin-autotooltip__default plugin-autotooltip_bigPreimplantation: in mammals, a preimplantation embryo is an embryo that has not yet attached to the uterus. embryo is crucial for the mouse geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. technology.

In order to make a directed genetic change in a specific mouse geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) we exploit homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species.. However, this is much harder to do in mammalian cells than bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. and 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 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, when a linear fragment of dsDNAplugin-autotooltip__default plugin-autotooltip_bigDouble-stranded DNA (dsDNA): DNA that consists of two complementary strands of ssDNA paired together via hydrogen bonds between the nitrogenous bases G, A, T, and C. is introduced into a 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 cell, that DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. is integrated into the 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 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 homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species. about 90% of the time, such that the incoming DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. fragment replaces the endogenous geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) on the chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins.. In mammalian cells, DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. that is introduced into a cell almost always integrates at a non-homologous site (see Chap. 15), and the frequency of homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species. at the endogenous locusplugin-autotooltip__default plugin-autotooltip_bigLocus (plural form: loci): a physical location of a gene; often used as a synonym for a gene. is very low - about 10^-3 to 10^-5 (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:

1. look through thousands of independent integration events; and
2. a way to be able to identify the specific integration event we want, namely an integration event that took place by homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species..

The first crucial technological advancement for creating mouse knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. was being able to grow the totipotentplugin-autotooltip__default plugin-autotooltip_bigTotipotent: used to describe stem cells that can potentially differentiate into all cell types of an organism. cells from preimplantationplugin-autotooltip__default plugin-autotooltip_bigPreimplantation: in mammals, a preimplantation embryo is an embryo that has not yet attached to the uterus. embryos in culture in the lab; these cells are called mouse embryonic stem cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. (ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish.). Cells from the inner cell mass of a preimplantationplugin-autotooltip__default plugin-autotooltip_bigPreimplantation: in mammals, a preimplantation embryo is an embryo that has not yet attached to the uterus. embryo at the blastocystplugin-autotooltip__default plugin-autotooltip_bigBlastocyst: an early stage embryo from which embryonic stem cells are derived. stage can be removed and cultured in a dish without the cells losing their totipotency (Fig. 2); even after being cultured in the lab for many years these cells could still be introduced back into a preimplantationplugin-autotooltip__default plugin-autotooltip_bigPreimplantation: in mammals, a preimplantation embryo is an embryo that has not yet attached to the uterus. embryo and go on to make all the tissues of a mouse.

This gave scientists an opportunity to genetically manipulate ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. in a Petri dishplugin-autotooltip__default plugin-autotooltip_bigPetri dish: a round dish, usually 5-10 cm in diameter, that can contain growth media to grow cells in vitro., in an analogous way as we might genetically manipulate 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 or E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). cells in a Petri dishplugin-autotooltip__default plugin-autotooltip_bigPetri dish: a round dish, usually 5-10 cm in diameter, that can contain growth media to grow cells in vitro.. Importantly, this further gives scientists the opportunity to isolate clonesplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of as coloniesplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of and also the ability to grow a large number of cells to screenplugin-autotooltip__default plugin-autotooltip_bigScreen: a screen is a process through which a researcher looks through a population of individuals in an attempt to find rare individuals with certain phenotypes, usually with no obvious way to enrich for the rare individuals. contrast to a selection. for rare events such as homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species. (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 cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. (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 totipotentplugin-autotooltip__default plugin-autotooltip_bigTotipotent: used to describe stem cells that can potentially differentiate into all cell types of an organism. ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish..

The second crucial technological advancement for creating mouse knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. was the invention of a clever scheme to selectplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a for ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. that integrate DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. via homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species.. Recall that we first introduced the concept of screensplugin-autotooltip__default plugin-autotooltip_bigScreen: a screen is a process through which a researcher looks through a population of individuals in an attempt to find rare individuals with certain phenotypes, usually with no obvious way to enrich for the rare individuals. contrast to a selection. and selectionsplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a in Chap. 08. A selectionplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a is simply a strategy for only allowing rare events you want to find to survive in an experiment. For generating mouse knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics., a double selectionplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a scheme is used - a positive selectionplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a and a negative selectionplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a (Fig. 3). We first create a targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele.. If we wanted to knock out geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) $X$, for instance, our target construct would have the following features:

• We need two DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. fragments with sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. that match portions of geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) $X$. Usually we will pick one fragment that is near the start of geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) $X$, and another fragment that is some distance downstreamplugin-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 from the first fragment. We can obtain these DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. fragments in a variety of ways, but 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. is probably the easiest way to do this. We call these two fragments the left homology arm and the right homology arm (sometimes just the left arm and right arm). Note that in Chap. 15, we discussed 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 and mentioned that 30-50 bp is sufficient for homology armsplugin-autotooltip__default plugin-autotooltip_bigHomology arm: a region of a targeting construct used to make knockout alleles that matches the DNA sequence of the endogenous locus. in a 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 knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. experiment. In mouse ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish., the homology armsplugin-autotooltip__default plugin-autotooltip_bigHomology arm: a region of a targeting construct used to make knockout alleles that matches the DNA sequence of the endogenous locus. must be much longer - usually at least around 1000 bp.
• In between these two fragments we will insert a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) called $neo^R$ (we also include all regulatory sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. needed for $neo^R$ to be 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 ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish.). ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. expressing $neo^R$ will be resistant to a drug called G-418plugin-autotooltip__default plugin-autotooltip_bigG-418: a drug that kills eukaryotic cells. A gene called $neo^R$ confers resistance to G-418.. Wildtypeplugin-autotooltip__default plugin-autotooltip_bigWildtype: a reference strain of an organism that scientists operationally define as “normal” to which mutants are compared. Not to be confused with wild organisms. ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. will be killed by G-418plugin-autotooltip__default plugin-autotooltip_bigG-418: a drug that kills eukaryotic cells. A gene called $neo^R$ confers resistance to G-418..
• To the “right” of the right homology arm, we will add another geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) called $tk^{HSV}$. This is a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) from herpes simplex virus (HSV) that codes for an enzymeplugin-autotooltip__default plugin-autotooltip_bigEnzyme: a macromolecule, usually a protein (but sometimes an RNA), that functions as a catalyst of some kind of biochemical reaction. called thymidine kinase. (We also include all regulatory sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. needed for $tk^{HSV}$ to be 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 ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish..) ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. that express $tk^{HSV}$ do not die unless they are exposed to the drug ganciclovirplugin-autotooltip__default plugin-autotooltip_bigGanciclovir: a drug (technically, a prodrug) that is harmless to eukaryotic cells but causes cells to die if it is acted upon by a gene called $tk^{HSV}$ from herpes simplex virus 1..

Using this targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele., we can selectplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a for homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species. in three steps.

• First, we transfect our targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. into ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish.. Instead of CaCl₂ or LiCl treatment we used to make E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). and 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 cell competent, we instead use a technology called electroporationplugin-autotooltip__default plugin-autotooltip_bigEletroporation: an experimental technique that can be used on both prokaryotic and eukaryotic cells to introduce foreign DNA into cells without using chemicals like LiCl or CaCl2. where cells are briefly shocked with a high-voltage pulse of electricity to make their membranes porous so that they can take up foreign DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth.¹⁾.
• Second, we use G-418plugin-autotooltip__default plugin-autotooltip_bigG-418: a drug that kills eukaryotic cells. A gene called $neo^R$ confers resistance to G-418. to positively selectplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:
  
  In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a for all ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. that have taken up our targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele.; all ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. that did not take up the targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. will die (another way we say this is that $neo^R$-expressing cells are selected for). At this point, the targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. is randomly integrated in most surviving cells, and homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species. at geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) $X$ has only occurred in rare instances.
• Third, we use ganciclovirplugin-autotooltip__default plugin-autotooltip_bigGanciclovir: a drug (technically, a prodrug) that is harmless to eukaryotic cells but causes cells to die if it is acted upon by a gene called $tk^{HSV}$ from herpes simplex virus 1. to treat the ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. that survived the second step. Because homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species. depends on the left and right homology armsplugin-autotooltip__default plugin-autotooltip_bigHomology arm: a region of a targeting construct used to make knockout alleles that matches the DNA sequence of the endogenous locus. and because $tk^{HSV}$ is outside of the homology armsplugin-autotooltip__default plugin-autotooltip_bigHomology arm: a region of a targeting construct used to make knockout alleles that matches the DNA sequence of the endogenous locus., the only ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. that can survive this step are ones that have undergone homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species. via the homology armsplugin-autotooltip__default plugin-autotooltip_bigHomology arm: a region of a targeting construct used to make knockout alleles that matches the DNA sequence of the endogenous locus. and excluded the $tk^{HSV}$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) from the chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins.. ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. in which the targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. has randomly 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 will include the $tk^{HSV}$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) and will undergo negative selectionplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:
  
  In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a (another way we say this is that $tk^{HSV}$-expressing cells will be selected against).

ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. that survive this multistep process now have a significant portion of geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) $X$ replaced with the $neo^R$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) - you have knocked out geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) $X$ in ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. (Figs. 3 and 4).

The next step is to create a mouse in which every cell in the mouse contains the genetic alteration you just created in ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish.. The first thing to note is that the ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. in which we knocked out geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) X are from a mouse strainplugin-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 that has gray fur. We will inject these ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. into a new blastocystplugin-autotooltip__default plugin-autotooltip_bigBlastocyst: an early stage embryo from which embryonic stem cells are derived. embryo that comes from a mouse strainplugin-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 that has white fur (Fig. 4). 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 chimerasplugin-autotooltip__default plugin-autotooltip_bigChimera: In the context of multicellular animals, a chimera is an individual that contains cells derived from multiple parents. In a general sense, “chimera” means something with a mixed constitution. In Greek mythology, a chimera is a monster that has a lion's head, a goat's body, and a serpent's tail. See also. We then take male chimericplugin-autotooltip__default plugin-autotooltip_bigChimera: In the context of multicellular animals, a chimera is an individual that contains cells derived from multiple parents. In a general sense, “chimera” means something with a mixed constitution. In Greek mythology, a chimera is a monster that has a lion's head, a goat's body, and a serpent's tail. See also pups and breed them to white females. Since the gray fur phenotypeplugin-autotooltip__default plugin-autotooltip_bigPhenotype: an observable feature or property of an organism. is 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., any chimericplugin-autotooltip__default plugin-autotooltip_bigChimera: In the context of multicellular animals, a chimera is an individual that contains cells derived from multiple parents. In a general sense, “chimera” means something with a mixed constitution. In Greek mythology, a chimera is a monster that has a lion's head, a goat's body, and a serpent's tail. See also father that has germ cellsplugin-autotooltip__default plugin-autotooltip_bigGamete: a specialized (usually haploid) cell used for sexual reproduction. Eggs (oocytes) and sperm are gametes. that formed from our modified ES cellsplugin-autotooltip__default plugin-autotooltip_bigEmbryonic stem cell: totipotent stem cells derived from preimplantion embryos that can be grown in vitro in a Petri dish. will produce pups with all gray fur; these pups are then 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. for a knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. 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 (Fig. 5).

We can apply the general strategy for making 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. described above to create mice that contain a knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. 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. of the mouse α-globin geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) and the mouse β-globin geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-). Together with the 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. for expressing human α-globin and human β-globin in mice, we generated in Chapter 15, we now have a way of creating a humanizedplugin-autotooltip__default plugin-autotooltip_bigHumanized: in genetics, a humanized organism is a genetically modified organism that has had one or more of its genes replaced with human versions of those genes, in order to create an animal model with which to study human genes in vivo. mouse - that is, a mouse that only expresses human hemoglobin. See Questions and exercises below.

Some final notes on our story: this humanizedplugin-autotooltip__default plugin-autotooltip_bigHumanized: in genetics, a humanized organism is a genetically modified organism that has had one or more of its genes replaced with human versions of those genes, in order to create an animal model with which to study human genes in vivo. 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 geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) therapy approaches to treating the disease. Although 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. therapies have rendered some of these treatments obsolete, we use this story to illustrate the power of 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 as a general strategy to study geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) function or genetic diseases.

We also note that while this chapter discusses the technique for how to make 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 mice, the idea of “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”, i.e., knocking out a specific geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) of interest (as opposed to randomly mutagenizing genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) and looking for interesting mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference.) is a general strategy for studying geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of 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. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs)., 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, or Drosophilaplugin-autotooltip__default plugin-autotooltip_bigDrosophila melanogaster: a fruit fly species used in genetics research.), although the precise technical details of how knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. can be generated for each organism will of course be somewhat different. A new tool developed around 2012 called CRISPRplugin-autotooltip__default plugin-autotooltip_bigCRISPR: short for clustered regularly interspaced short palindromic repeats. In bacteria, CRISPR is a form of innate immunity that bacterial cells use as a defense against bacteriophage. The CRISPR system consists of a protein typically called Cas9 and two non-coding RNAs called gRNA and tracrRNA. The (briefly discussed in Chapter 17) has made this geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. approach for studying geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) function much more accessible to organisms other than the traditional model organisms.

Conceptual question: How many different genetic parents does the chimericplugin-autotooltip__default plugin-autotooltip_bigChimera: In the context of multicellular animals, a chimera is an individual that contains cells derived from multiple parents. In a general sense, “chimera” means something with a mixed constitution. In Greek mythology, a chimera is a monster that has a lion's head, a goat's body, and a serpent's tail. See also mouse shown in Fig. 5 above have?

Conceptual question: What will the genotypeplugin-autotooltip__default plugin-autotooltip_bigGenotype: the combination of alleles within an organism or strain. When used as a verb, it means to determine the genotype experimentally. for geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) $X$ be for the all-gray founderplugin-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. pups described in Fig. 5?

Exercise 1. (Challenge Question) Assuming that all the mouse genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) and 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. described from Chap. 15 and this chapter are unlinkedplugin-autotooltip__default plugin-autotooltip_bigLinkage: two loci are linked to each other if they are less than 50 m.u. apart. Two loci are unlinked if they are either (1) greater than 50 m.u. apart on the same chromosome, or; (2) are on separate chromosomes. to each other and that everything is autosomalplugin-autotooltip__default plugin-autotooltip_bigAutosome: any chromosome that is not a sex chromosome. (i.e., nothing is sex-linkedplugin-autotooltip__default plugin-autotooltip_bigSex linkage: a gene is said to be sex-linked if it maps to a sex chromosome.), how could you breed a humanizedplugin-autotooltip__default plugin-autotooltip_bigHumanized: in genetics, a humanized organism is a genetically modified organism that has had one or more of its genes replaced with human versions of those genes, in order to create an animal model with which to study human genes in vivo. mouse that only 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. human hemoglobin? You start out with individual knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. and 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. lines. Let's also assume you have an easy way to genotypeplugin-autotooltip__default plugin-autotooltip_bigGenotype: the combination of alleles within an organism or strain. When used as a verb, it means to determine the genotype experimentally. everything (usually in these kinds of experiments you would use 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. for genotyping). Keep in mind that the globin genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) are essential, and that the knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. are homozygousplugin-autotooltip__default plugin-autotooltip_bigHomozygous: a state for a diploid organism wherein the two alleles for a gene are identical to each other. lethal. In each step, what is the likelihood of getting progenyplugin-autotooltip__defaultProgeny: a synonym for offspring. that have the genotypesplugin-autotooltip__default plugin-autotooltip_bigGenotype: the combination of alleles within an organism or strain. When used as a verb, it means to determine the genotype experimentally. you need?