TNBGGA: The No Bullshit Guide to Genetic Analysis

Geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knock-ins are a variation of the classic 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. we saw in Chap. 16. In this method, instead of replacing an endogenous geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) with something like $neo^R$, we replace it with a modified version of the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-). For instance, instead of going through the complex 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./knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. combination strategy described in Chapters 15-16, you could in principle simply replace the mouse globin genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) with knock-ins of the human globin genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-). (When the studies described in Chapters 15-16 were originally done, the knock-inplugin-autotooltip__default plugin-autotooltip_bigKnock-in: a reverse genetic experimental approach that replaces an endogenous copy of a gene with a modified one. technique had not yet been invented.) This greatly reduces the complexity of the experimental design of the globin geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) project. Furthermore, the level of 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 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. is often difficult to control precisely because 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. often use 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 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 are removed from their normal chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. context (the 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. used may be missing some important enhancerplugin-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. 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., for instance). By comparison, a knock-inplugin-autotooltip__default plugin-autotooltip_bigKnock-in: a reverse genetic experimental approach that replaces an endogenous copy of a gene with a modified one. 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. usually will 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. at very similar levels as the 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. 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., because all the normal enhancerplugin-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. 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 regulate 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. are still there.

To understand how geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knock-ins work, we must first discuss a site-specific recombinationplugin-autotooltip__default plugin-autotooltip_bigRecombination: Recombination can have slightly different meanings depending on context:

* In the context of genetic crosses (usually a dihybrid cross or a test cross), recombination refers to the phenomena where the phenotype of the F2 offspring is different than either parent (P generation). $lox$ system such as the Creplugin-autotooltip__default plugin-autotooltip_bigCre-$lox$ system: A site-specific DNA recombination system derived from bacteriophage P1. The enzyme Cre recombinase will drive DNA recombination between 34 bp-long $loxP$ sequences. Very commonly used in various reverse genetic experiments.-$lox$ system from bacteriophageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. P1 (Fig. 1). $loxP$ is a 34 bp DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequenceplugin-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. from 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 of bacteriophageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. P1. Normally, 34 bp is not long enough to drive 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 mice. However, a bacteriophageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. P1 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 Creplugin-autotooltip__default plugin-autotooltip_bigCre-$lox$ system: A site-specific DNA recombination system derived from bacteriophage P1. The enzyme Cre recombinase will drive DNA recombination between 34 bp-long $loxP$ sequences. Very commonly used in various reverse genetic experiments. (also called Cre recombinaseplugin-autotooltip__default plugin-autotooltip_bigCre-$lox$ system: A site-specific DNA recombination system derived from bacteriophage P1. The enzyme Cre recombinase will drive DNA recombination between 34 bp-long $loxP$ sequences. Very commonly used in various reverse genetic experiments.) can cause DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. recombinationplugin-autotooltip__default plugin-autotooltip_bigRecombination: Recombination can have slightly different meanings depending on context:

* In the context of genetic crosses (usually a dihybrid cross or a test cross), recombination refers to the phenomena where the phenotype of the F2 offspring is different than either parent (P generation). $lox$ (essentially crossing overplugin-autotooltip__default plugin-autotooltip_bigCrossing over: an event where non-sister chromatids exchange material with each other during meiosis I.) to occur between two different $loxP$ sites. This results in the 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. between the $loxP$ sites being deleted, leaving behind a single $loxP$ site as a recombinationplugin-autotooltip__default plugin-autotooltip_bigRecombination: Recombination can have slightly different meanings depending on context:

* In the context of genetic crosses (usually a dihybrid cross or a test cross), recombination refers to the phenomena where the phenotype of the F2 offspring is different than either parent (P generation). $lox$ scar.

To generate a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knock-inplugin-autotooltip__default plugin-autotooltip_bigKnock-in: a reverse genetic experimental approach that replaces an endogenous copy of a gene with a modified one., you would usually follow the normal 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. experimental approach, with a few modifications. For example, let's say you want to modify a codonplugin-autotooltip__default plugin-autotooltip_bigCodon: a three nucleotide sequence that is read by the ribosome and specifies an amino acid that is added to a growing poplypeptide chain based on the genetic code. in exonplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons. 2 of a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (Fig. 2). Here is how you might design your targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele.:

• You would cloneplugin-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 (probably using 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.) DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. 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. from exonplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons. 1, intronplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns. 1, and exonplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons. 2.
• You would insert $neo^R$ into intronplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns. 1, flanked by $loxP$ sites on either side.
• You would engineer the 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. you want into the appropriate sequenceplugin-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. in exonplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons. 2.
• $tk^{HSV}$ would go after exonplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons. 2.

This targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. can be used to generate a “knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics.”, but since $neo^R$ is located in an intronplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns., it doesn't really disrupt the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) at all. This targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. will integrate 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. using 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. from exonplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons. 1 and exonplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons. 2 in the targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele., thereby bringing in your engineered 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. to the correct chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. location in 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.. Once you have targeted 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., you can remove the $neo^R$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) from the intronplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns. by expressing Creplugin-autotooltip__default plugin-autotooltip_bigCre-$lox$ system: A site-specific DNA recombination system derived from bacteriophage P1. The enzyme Cre recombinase will drive DNA recombination between 34 bp-long $loxP$ sequences. Very commonly used in various reverse genetic experiments. in 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. before creating 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 mice, leaving behind a $loxP$ scar in the intronplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns.. This is usually done with a separate 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. that expresses Creplugin-autotooltip__default plugin-autotooltip_bigCre-$lox$ system: A site-specific DNA recombination system derived from bacteriophage P1. The enzyme Cre recombinase will drive DNA recombination between 34 bp-long $loxP$ sequences. Very commonly used in various reverse genetic experiments..

Conditional knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. are a variation of the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knock-inplugin-autotooltip__default plugin-autotooltip_bigKnock-in: a reverse genetic experimental approach that replaces an endogenous copy of a gene with a modified one. method (Fig. 3). A conditional knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. is a 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. that only occurs under certain conditions, such as in specific cell types or at specific developmental time points. To create a conditional knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics., you would use the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knock-inplugin-autotooltip__default plugin-autotooltip_bigKnock-in: a reverse genetic experimental approach that replaces an endogenous copy of a gene with a modified one. method above to add two $loxP$ sites in two different intronsplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns. of a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) you wanted to knock out. Since the $loxP$ sites are in intronsplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns., they typically will not affect the function of the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-); this 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. usually will be indistinguishable from 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.. If you can somehow express Creplugin-autotooltip__default plugin-autotooltip_bigCre-$lox$ system: A site-specific DNA recombination system derived from bacteriophage P1. The enzyme Cre recombinase will drive DNA recombination between 34 bp-long $loxP$ sequences. Very commonly used in various reverse genetic experiments. in these cells, then Creplugin-autotooltip__default plugin-autotooltip_bigCre-$lox$ system: A site-specific DNA recombination system derived from bacteriophage P1. The enzyme Cre recombinase will drive DNA recombination between 34 bp-long $loxP$ sequences. Very commonly used in various reverse genetic experiments. will cause recombinationplugin-autotooltip__default plugin-autotooltip_bigRecombination: Recombination can have slightly different meanings depending on context:

* In the context of genetic crosses (usually a dihybrid cross or a test cross), recombination refers to the phenomena where the phenotype of the F2 offspring is different than either parent (P generation). $lox$ to occur between the two $loxP$ sites, which will delete all the exonsplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons. in between. This usually results in a frameshiftplugin-autotooltip__default plugin-autotooltip_bigFrameshift mutation: an indel mutation in which the net number of base pairs added or removed is not a multiple of three. This disrupts the open reading frame and drastically alters the amino acid sequence past the site of the indel; this usually results in a premature stop codon. 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., and frameshiftsplugin-autotooltip__default plugin-autotooltip_bigFrameshift mutation: an indel mutation in which the net number of base pairs added or removed is not a multiple of three. This disrupts the open reading frame and drastically alters the amino acid sequence past the site of the indel; this usually results in a premature stop codon. usually result in a nullplugin-autotooltip__default plugin-autotooltip_bigAmorphic mutation: assuming a wildtype allele has 100% gene function, an amorphic mutation is a mutant allele that has 0% gene function. Also called a null mutation or a complete loss of function mutation. 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 conditional knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. experiments, Creplugin-autotooltip__default plugin-autotooltip_bigCre-$lox$ system: A site-specific DNA recombination system derived from bacteriophage P1. The enzyme Cre recombinase will drive DNA recombination between 34 bp-long $loxP$ sequences. Very commonly used in various reverse genetic experiments. is usually 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. as a 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., and there are other tricks that can be done to express Creplugin-autotooltip__default plugin-autotooltip_bigCre-$lox$ system: A site-specific DNA recombination system derived from bacteriophage P1. The enzyme Cre recombinase will drive DNA recombination between 34 bp-long $loxP$ sequences. Very commonly used in various reverse genetic experiments. only in specific cells, such as using viral vectors, or to only express Creplugin-autotooltip__default plugin-autotooltip_bigCre-$lox$ system: A site-specific DNA recombination system derived from bacteriophage P1. The enzyme Cre recombinase will drive DNA recombination between 34 bp-long $loxP$ sequences. Very commonly used in various reverse genetic experiments. under the control of an inducibleplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive. 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 that depends on an external inducerplugin-autotooltip__default plugin-autotooltip_bigInducer: a molecule that triggers the expression of a gene or genes. Examples in E. coli include lactose and IPTG, which induce expression of the Lac operon in E. coli, and maltose, which induces expression of the Mal operon. An example in yeast is galactose, which induces expression of Gal genes., or some combination thereof.

Conditional knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. are useful for studying genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that 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. in multiple tissues, but you only wish to study the effect in one kind of tissue. For instance, let's say you are studying geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) $X$, which 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. in both brain and liver tissue. When you knock out geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) $X$ using the standard approach, you find that the mice die as embryos due to a brain defect during embryogenesis. However, you are interested in the role of geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) $X$ in the livers of adult mice. You can generate a conditional knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. of geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) $X$ where it is only knocked out in adult livers but remains functional in embryonic brains so that they can be born and develop into adults that you can then study for liver function. One way to achieve this is to find a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that is 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. in adult livers, cloneplugin-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 the 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 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 regulate 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. in adult liver cells, and generate a 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. that expresses Creplugin-autotooltip__default plugin-autotooltip_bigCre-$lox$ system: A site-specific DNA recombination system derived from bacteriophage P1. The enzyme Cre recombinase will drive DNA recombination between 34 bp-long $loxP$ sequences. Very commonly used in various reverse genetic experiments. under the control of this regulatory sequenceplugin-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.. You can then generate a floxed alleleplugin-autotooltip__default plugin-autotooltip_bigFloxed allele: an allele that has been engineered to contain two $loxP$ sites, such that when Cre recombinase is expressed, the allele undergoes recombination and deletes the sequence in between the $loxP$ sites. (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. with flanking $loxP$ sites) of geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) $X$ and breed mice that carry this 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. together with your liver-specific Creplugin-autotooltip__default plugin-autotooltip_bigCre-$lox$ system: A site-specific DNA recombination system derived from bacteriophage P1. The enzyme Cre recombinase will drive DNA recombination between 34 bp-long $loxP$ sequences. Very commonly used in various reverse genetic experiments. 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..

As we learned in Chap. 16, 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. occurs only at low frequencies in mammalian cells – this is why it was necessary to develop technologies to culture 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 vitroplugin-autotooltip__default plugin-autotooltip_bigIn vitro: taking place outside of a living organism, usually in a test tube or Petri dish., so that we could examine large numbers of 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. to look for rare events. It turns out that 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. increases enormously if there is a double-stranded break in the DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. near the site you wish to have 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.. 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/Cas9plugin-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 (Fig. 4) is 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. usually isolated from the bacteriumplugin-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. Streptococcus pyogenes that can be used to cleave 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. to generate a double-stranded break. However, unlike restriction enzymesplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. that only cut short defined palindromic 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. (Chap. 9), it is possible to modify 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/Cas9plugin-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 such that we can essentially make it cut almost any unique DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequenceplugin-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. we wish. This is possible because 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/Cas9plugin-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 uses a guide RNAplugin-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 (gRNAplugin-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) as part of its 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. structure and the gRNAplugin-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 sequenceplugin-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. is what guides it to its target DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. through RNAplugin-autotooltip__default plugin-autotooltip_bigRNA sequencing (RNAseq): an experimental technique that sequences all the RNAs in a sample. It is based off of converting RNAs into cDNAs with reverse transcriptase, followed by Illumina sequencing./DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. base pairingplugin-autotooltip__default plugin-autotooltip_bigBase pair: a term used to describe how nitrogenous bases (G, A, T/U, and C) in nucleic acids interact with each other via hydrogen bonds to form double-stranded molecules (including dsDNA, dsRNA, and DNA/RNA hybrids). G always pairs with C, and T/U always pairs with A.. It is relatively easy to modify this RNA sequenceplugin-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. so that 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/Cas9plugin-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 will target the DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequenceplugin-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. of a specific geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) you are interested in. When 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 cleaved in live cells, repair mechanisms will rejoin the cleaved DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth.; however, errors often occur during this process, and indelplugin-autotooltip__default plugin-autotooltip_bigIndel mutation: “Indel” is a portmanteau of “insertion” and “deletion”. Indels are mutations that insert or delete base pairs from a gene sequence. 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. usually occur and therefore frameshiftsplugin-autotooltip__default plugin-autotooltip_bigFrameshift mutation: an indel mutation in which the net number of base pairs added or removed is not a multiple of three. This disrupts the open reading frame and drastically alters the amino acid sequence past the site of the indel; this usually results in a premature stop codon., which usually creates nullplugin-autotooltip__default plugin-autotooltip_bigAmorphic mutation: assuming a wildtype allele has 100% gene function, an amorphic mutation is a mutant allele that has 0% gene function. Also called a null mutation or a complete loss of function mutation. allelesplugin-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 a targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. is also present, 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. will occur at a very high frequency; this allows for creation of knock-inplugin-autotooltip__default plugin-autotooltip_bigKnock-in: a reverse genetic experimental approach that replaces an endogenous copy of a gene with a modified one. allelesplugin-autotooltip__default plugin-autotooltip_bigAllele: a version of a gene. Alleles of a gene are different if they have differences in their DNA sequence. without necessarily using the Creplugin-autotooltip__default plugin-autotooltip_bigCre-$lox$ system: A site-specific DNA recombination system derived from bacteriophage P1. The enzyme Cre recombinase will drive DNA recombination between 34 bp-long $loxP$ sequences. Very commonly used in various reverse genetic experiments.-$lox$-based strategy described above.

Using 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/Cas9plugin-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, it is possible to create knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. and knock-ins in other model organisms such as C. elegansplugin-autotooltip__default plugin-autotooltip_bigCaenorhabditis elegans: a non-parasitic non-pathogenic nematode species used as a model organism in genetics researhc. and zebrafish where culturing 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. is not possible, and it is even possible to do it directly in somatic tissue without having to generate knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics./knock-inplugin-autotooltip__default plugin-autotooltip_bigKnock-in: a reverse genetic experimental approach that replaces an endogenous copy of a gene with a modified one. mice. 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/Cas9plugin-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-generated 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. breaks make 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. so efficient that it has largely replaced the traditional method of generating knock-ins and knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. using the 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 method. 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/Cas9plugin-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 can even be used for geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) therapy in humans. If there is a 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 patient, 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/Cas9plugin-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 can potentially be used to modify it so that it no longer has deleterious effects.

Some viruses can be modified to deliver genetic material of any desired sequenceplugin-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. into cells. Traditionally, viral vectorsplugin-autotooltip__default plugin-autotooltip_bigVector: in molecular genetics, a vector is a genetic element (usually an episome of sort such as a plasmid but can be something like a virus) that is used to clone and/or transfer genes into and out of cells. such as adeno-associated virusesplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. (AAVsplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”.) or lentiviruses (a type of retrovirus) have been used to deliver modified genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) into cultured cells in an analogous approach as generating 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.. These types of viral vectorsplugin-autotooltip__default plugin-autotooltip_bigVector: in molecular genetics, a vector is a genetic element (usually an episome of sort such as a plasmid but can be something like a virus) that is used to clone and/or transfer genes into and out of cells. can also be used to deliver 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. into live animals without using microinjection as described in Chap. 15. A modern experimental approach to knock out geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) function that is much faster than generating knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. or knock-ins with 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. is to use a viral vectorplugin-autotooltip__default plugin-autotooltip_bigVector: in molecular genetics, a vector is a genetic element (usually an episome of sort such as a plasmid but can be something like a virus) that is used to clone and/or transfer genes into and out of cells. to deliver genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) coding for a modified 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/Cas9plugin-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 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. directly into live tissue in an animal. This allows you to analyze loss of functionplugin-autotooltip__default plugin-autotooltip_bigLoss of function: a general term used to describe mutant alleles that have less activity than wildtype. Amorphic and hypomorphic mutations are loss of function mutations. of a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) in live tissue in a live animal without having to go through the trouble of creating a knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. mouse (although depending on your needs, a knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. mouse can also be very valuable).

Let's look at AAVsplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. a bit more closely (Fig. 5).

Naturally occurring AAVsplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. are small nonpathogenic parvoviruses. The AAVplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. 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 is just under 5 kbp long and is made of ssDNAplugin-autotooltip__default plugin-autotooltip_bigSingle-stranded DNA (ssDNA): a polymerized chain of deoxyribonucleotides that is not paired with a complementary polymer. Usually formed by denaturing dsDNA with heat or other methods. instead of the more typical 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.. The ends of the ssDNAplugin-autotooltip__default plugin-autotooltip_bigSingle-stranded DNA (ssDNA): a polymerized chain of deoxyribonucleotides that is not paired with a complementary polymer. Usually formed by denaturing dsDNA with heat or other methods. 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 form structures called inverted terminal repeatsplugin-autotooltip__default plugin-autotooltip_bigTerminal repeats: DNA sequences found at the ends of some transposons and viruses. They are required for transposon or viral function. (ITRs; Fig. 5) that are required for replicating and packaging viral ssDNAs into new viral capsidsplugin-autotooltip__default plugin-autotooltip_bigCapsid: the outer shell of some viruses made from various viral proteins.. AAVsplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. cannot successfully replicate in cells by themselves because they depend on 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. provided by another kind of virus (usually an adenovirus or herpesvirus) to produce new AAVplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. viral particles after infection. For instance, three 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. from adenovirus, E2A, E4, and VA, are required for AAVplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. replicationplugin-autotooltip__default plugin-autotooltip_bigDNA replication: usually the process of starting with a dsDNA molecule and ending with two identical copies of that dsDNA molecule. In most cases, “replication” implies DNA replication..

To produce recombinantplugin-autotooltip__default plugin-autotooltip_bigRecombinant: (adj.) Describing something that has undergone recombination, e.g., recombinant DNA or recombinant offspring. “Non-parental” is a synonym when referring to organisms. (n.) Something that has undergone recombination, e.g., “This fly is a recombinant.” AAVsplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. (rAAVsplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”.; Fig. 6) as a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) delivery vectorplugin-autotooltip__default plugin-autotooltip_bigVector: in molecular genetics, a vector is a genetic element (usually an episome of sort such as a plasmid but can be something like a virus) that is used to clone and/or transfer genes into and out of cells. for research or therapeutic purposes, you would need to construct three separate plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. in vitroplugin-autotooltip__default plugin-autotooltip_bigIn vitro: taking place outside of a living organism, usually in a test tube or Petri dish.:

• A plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. that expresses the Rep and Cap 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 are normally encoded by the AAVplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. 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 (purple in Fig. 6);
• A second plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. that contains 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. of interest you are trying to deliver into cells (red in Fig. 6);
• A third plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. that expresses the essential E2A, E4, and VA genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) from adenovirus (green in Fig. 6)

You would then introduce all three plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. into cultured cells (such as HEK293 cells, a human embryonic kidney cell 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). The purple and green plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors., together with the HEK293 host cell 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, provide all the 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. needed to replicate and package newly replicated viral ssDNAs. However, only DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. with ITRs will be packaged. This means that the plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. containing AAVplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. and adenoviruses will not be packaged (they won't even be replicated) - only the ssDNAplugin-autotooltip__default plugin-autotooltip_bigSingle-stranded DNA (ssDNA): a polymerized chain of deoxyribonucleotides that is not paired with a complementary polymer. Usually formed by denaturing dsDNA with heat or other methods. with the ITRs, and therefore 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., will be packaged into new viral capsidsplugin-autotooltip__default plugin-autotooltip_bigCapsid: the outer shell of some viruses made from various viral proteins.. You can harvest the rAAVsplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. from the media of the HEK293 cell culture.

The rAAVsplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. can then be used to deliver your geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) payload into target cells through infection. Different serotypesplugin-autotooltip__default plugin-autotooltip_bigSerotype: in the context of Adeno-associated viruses, a serotype is a variant of the virus with different capsid proteins. AAVs of different serotypes can infect different cell types. (or variations) of AAVplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. capsidplugin-autotooltip__default plugin-autotooltip_bigCapsid: the outer shell of some viruses made from various viral proteins. 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. can be used to target your rAAVsplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. to different tissue types (Table 1). These rAAVsplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. cannot produce new rAAVplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. capsidsplugin-autotooltip__default plugin-autotooltip_bigCapsid: the outer shell of some viruses made from various viral proteins. after infection of target tissue because they do not encode the essential AAVplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) Rep, Cap, and AAP. In principle, this makes rAAVsplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. ideal for a vehicle for geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) therapy. One drawback of AAVsplugin-autotooltip__default plugin-autotooltip_bigAdeno-associated virus (AAV): a small eukaryotic virus that is modified and used for research and therapeutic purposes as a gene delivery vector. rAAV stands for “recombinant AAV”. is that there is a size limit to their DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. capacity - around 5 kbp is the maximum payload size. There are some tricks that can be used to get around this size limit, but it can be inconvenient if 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. you wish to deliver into cells is greater than 5 kbp in length.

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