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  1. Article ; Online: Getting around an Early Lethal Phenotype in Mice with Chimeras.

    Papaioannou, Virginia E / Behringer, Richard R

    Cold Spring Harbor protocols

    2024  Volume 2024, Issue 1, Page(s) pdb.over107979

    Abstract: The same gene can have many different functions in different places in the body and/or at different times in development and adult life. Often only one organ or one developmental stage is of particular interest to an investigator. If, however, lethality ... ...

    Abstract The same gene can have many different functions in different places in the body and/or at different times in development and adult life. Often only one organ or one developmental stage is of particular interest to an investigator. If, however, lethality or severe detrimental effects of a mutation prevent the study of the organ or stage of interest, there are a number of ways to circumvent an early effect. In this overview, we discuss one way of getting around an early lethal phenotype by using chimeras, a method that is also useful for studying the mutant cells in the context of a wild-type host as part of the phenotypic analysis. The composition of chimeras with respect to embryonic cell lineages can be controlled to some extent to produce lineage-restricted chimeras with, for example, mutant cells restricted to certain lineages. Depending on the site of action of the mutant gene, this could result in chimeric "rescue." Details of how to distinguish mutant cells from wild type, an essential part of any chimera experiment, are discussed as well as methods to genotype the chimeras with respect to both component cell types.
    MeSH term(s) Mice ; Animals ; Phenotype ; Genotype ; Cell Lineage
    Language English
    Publishing date 2024-01-02
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.over107979
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Mouse Gene-Targeting Strategies for Maximum Ease and Versatility.

    Papaioannou, Virginia E / Behringer, Richard R

    Cold Spring Harbor protocols

    2024  Volume 2024, Issue 1, Page(s) pdb.over107957

    Abstract: Well-planned strategies are an essential prerequisite for any mutational analysis involving gene targeting. Consideration of the advantages or disadvantages of different methods will aid in the production of a final product that is both technically ... ...

    Abstract Well-planned strategies are an essential prerequisite for any mutational analysis involving gene targeting. Consideration of the advantages or disadvantages of different methods will aid in the production of a final product that is both technically feasible and versatile. Strategies for gene-targeting experiments in the mouse are discussed, including the rationale behind some of the common elements of gene-targeting vectors, such as homologous DNA and the use of different site-specific recombinases. We detail positive and negative selection as well as screening strategies for homologous recombination events in embryonic stem (ES) cells. For the planning stages of making different types of alleles, we first consider general strategies and then provide details specific to either homologous recombination in ES cells or making alleles by gene editing with CRISPR-Cas in preimplantation embryos. The types of alleles considered are null or knockout alleles, reporter gene knock-in alleles, point mutations, and conditional null alleles.
    MeSH term(s) Mice ; Animals ; Mice, Knockout ; Gene Targeting/methods ; Homologous Recombination ; Embryonic Stem Cells ; CRISPR-Cas Systems
    Language English
    Publishing date 2024-01-02
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.over107957
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Backcrossing to Generate a Congenic Mouse Strain.

    Papaioannou, Virginia E / Behringer, Richard R

    Cold Spring Harbor protocols

    2024  Volume 2024, Issue 1, Page(s) pdb.prot108039

    Abstract: Genetic background can have subtle or profound effects on mutant phenotypes, providing additional information regarding the function of the gene. If your mutation is maintained on one genetic background but you wish to analyze it on another, it is a ... ...

    Abstract Genetic background can have subtle or profound effects on mutant phenotypes, providing additional information regarding the function of the gene. If your mutation is maintained on one genetic background but you wish to analyze it on another, it is a simple matter to transfer the mutation to a recipient strain background by repeated backcrossing (introgression) as detailed in this protocol. The resulting strain is called a congenic strain, defined as a strain carrying the mutation within a segment of chromosome from the donor strain with the remainder of the genome from the recipient strain.
    MeSH term(s) Mice ; Animals ; Genome ; Phenotype ; Animals, Laboratory/genetics ; Chromosomes
    Language English
    Publishing date 2024-01-02
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.prot108039
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Recovering a Targeted Mutation in Mice from Embryonic Stem Cell Chimeras or CRISPR-Cas Founders.

    Papaioannou, Virginia E / Behringer, Richard R

    Cold Spring Harbor protocols

    2024  Volume 2024, Issue 1, Page(s) pdb.over107959

    Abstract: Following the production of chimeras from targeted embryonic stem (ES) cells or obtaining founders from CRISPR-Cas gene editing in preimplantation embryos, the desired targeted mutation must be recovered and established in the heterozygous state in a ... ...

    Abstract Following the production of chimeras from targeted embryonic stem (ES) cells or obtaining founders from CRISPR-Cas gene editing in preimplantation embryos, the desired targeted mutation must be recovered and established in the heterozygous state in a strain or stock of mice for further study. The breeding schemes for ES chimeras and CRISPR-Cas founders differ. For ES cell chimeras, we discuss the relative benefits of breeding from male or female chimeras. We discuss the importance of genetic background and provide practical advice for putting the mutation on inbred or outbred backgrounds or producing a coisogenic strain. For CRISPR-Cas founders, which will most likely be mosaic for different mutations, initial breeding strategies are discussed to maintain a desired genetic background at the same time as producing progeny to segregate different alleles. Strategies for testing the progeny to recognize indels, missense mutations, and knock-in mutations are discussed. In the event that ES cell chimeras or CRISPR-Cas founders produce no offspring or fail to transmit the mutant allele(s), there is a troubleshooting guide to pinpoint the problem. If heterozygous offspring from the chimeras or founders are normal, fertile, and of both sexes, the analysis of homozygous effects of the mutation can now begin; if not, possible dominant effects are considered.
    MeSH term(s) Mice ; Animals ; Male ; Female ; CRISPR-Cas Systems ; Mutation ; Gene Editing ; Embryonic Stem Cells ; Mutagenesis, Site-Directed
    Language English
    Publishing date 2024-01-02
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.over107959
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Phenotypic Analysis of Dominant Mutant Effects in Mice.

    Papaioannou, Virginia E / Behringer, Richard R

    Cold Spring Harbor protocols

    2024  Volume 2024, Issue 1, Page(s) pdb.over107978

    Abstract: Dominant effects of a mutation may show up at any time during a mutational analysis, including during the early stages of an embryonic stem (ES) cell gene targeting experiment. Here, we discuss the mechanisms of dominant and semidominant effects and how ... ...

    Abstract Dominant effects of a mutation may show up at any time during a mutational analysis, including during the early stages of an embryonic stem (ES) cell gene targeting experiment. Here, we discuss the mechanisms of dominant and semidominant effects and how they might appear if they show up in heterozygous ES cells, in ES cell chimeras, or in heterozygous progeny of chimeras. Similarly, dominant effects may be seen in mice heterozygous for CRISPR-Cas-targeted, -induced, or spontaneous mutations. If the dominant effects prevent the germline transmission of ES cells or cause fertility problems in heterozygotes, they can severely limit further analysis of the mutation. Ways to circumvent such reproductive problems are presented. The special case of imprinted genes, which may be functionally hemizygous and present a different phenotype when inherited from the mother than when inherited from the father, is discussed.
    MeSH term(s) Mice ; Animals ; Embryonic Stem Cells ; Mutation ; Phenotype
    Language English
    Publishing date 2024-01-02
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.over107978
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    Kategorien

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    Inter-library loan at ZB MED

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  6. Article ; Online: Necropsy Guide for the Collection of Tissues from Mice with or without Tumors.

    Papaioannou, Virginia E / Behringer, Richard R

    Cold Spring Harbor protocols

    2024  Volume 2024, Issue 1, Page(s) pdb.prot108097

    Abstract: Mice that die at any stage of a mutational analysis, whether during early life or during ageing or longitudinal studies such as tumor survival studies, can yield important information. This protocol provides a necropsy guide for the collection and ... ...

    Abstract Mice that die at any stage of a mutational analysis, whether during early life or during ageing or longitudinal studies such as tumor survival studies, can yield important information. This protocol provides a necropsy guide for the collection and processing of tissue samples to provide material for complete histological or immunostaining analysis.
    MeSH term(s) Mice ; Animals ; Neoplasms ; Autopsy/methods
    Language English
    Publishing date 2024-01-02
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.prot108097
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    Kategorien

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  7. Article ; Online: The "No Phenotype" Challenge in Analyzing Mutant Mice.

    Papaioannou, Virginia E / Behringer, Richard R

    Cold Spring Harbor protocols

    2024  Volume 2024, Issue 1, Page(s) pdb.over107977

    Abstract: If homozygous mutant mice survive to adulthood, are fertile, and have no visible phenotypes attributable to mutation of the relevant gene, there are a number of possible reasons why an effect of the mutation is not evident. Technical errors that might ... ...

    Abstract If homozygous mutant mice survive to adulthood, are fertile, and have no visible phenotypes attributable to mutation of the relevant gene, there are a number of possible reasons why an effect of the mutation is not evident. Technical errors that might have occurred during gene targeting or genotyping must first be eliminated. Variable penetrance of the mutation should be considered as well as the possibility of age-related or late-onset phenotypes, such as tumor formation or other pathologies. The gene expression pattern and nature of the protein product of the gene could provide clues. A number of simple tests can be applied to uncover cryptic phenotypes that are not easily seen on casual inspection (e.g., tests of the senses and of balance and coordination). Genetic and environmental challenges can be applied to overtly normal mutant mice to reveal deviations from normal.
    MeSH term(s) Mice ; Animals ; Phenotype ; Mutation ; Proteins/genetics
    Chemical Substances Proteins
    Language English
    Publishing date 2024-01-02
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.over107977
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    Kategorien

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  8. Article ; Online: Special Breeding Techniques for Use in Mouse Mutation Analysis.

    Papaioannou, Virginia E / Behringer, Richard R

    Cold Spring Harbor protocols

    2024  Volume 2024, Issue 1, Page(s) pdb.over107961

    Abstract: Certain specialized breeding techniques may come in handy during the analysis of a mutation in order to further understanding of the mutation and its interactions with other genes. Different mutant alleles of the gene in question might be available from ... ...

    Abstract Certain specialized breeding techniques may come in handy during the analysis of a mutation in order to further understanding of the mutation and its interactions with other genes. Different mutant alleles of the gene in question might be available from other sources or mutations with similar phenotypes could potentially be alleles. This could be determined by complementation testing. In the production of a conditional allele, retention of exogenous DNA in the allele could fortuitously disrupt a regulatory element and thus result in a hypomorphic allele, which can be simply tested by breeding. Mutations in different genes frequently affect the same organ, tissue, or cell type through genetic interactions. Common approaches to investigate and interpret genetic interactions are detailed here for gene families, in which there may be redundancy or genetic compensation of different genes, for genes that constitute different components of a biochemical pathway, for genes with overlapping expression patterns, and for unrelated genes that produce similar mutant phenotypes.
    MeSH term(s) Mice ; Animals ; Mutation ; Phenotype ; Alleles
    Language English
    Publishing date 2024-01-02
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.over107961
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  9. Article ; Online: Analysis of Mid- to Late-Gestation Phenotypes in Mice.

    Papaioannou, Virginia E / Behringer, Richard R

    Cold Spring Harbor protocols

    2024  Volume 2024, Issue 1, Page(s) pdb.over107973

    Abstract: Mid- to late gestation is characterized by tissue differentiation, maturation, organogenesis, and growth, and many mutant genes have detrimental effects during this phase of development. The outcome may be lethal before birth or may be compatible with ... ...

    Abstract Mid- to late gestation is characterized by tissue differentiation, maturation, organogenesis, and growth, and many mutant genes have detrimental effects during this phase of development. The outcome may be lethal before birth or may be compatible with life but result in birth defects. Some of the common causes of death during late gestation are hematopoietic defects, cardiovascular problems, and placental insufficiency. Many morphological abnormalities, lethal or not, can be investigated with gross and histological analyses or by visualization of the developing skeleton. Molecular characterization of mutant phenotypes, guided by the expression pattern of the mutant gene, can reveal disruptions in gene expression patterns of known developmental genes. Cell proliferation and cell death assays will reveal disruptions in cellular dynamics. Various modalities of 3D imaging of intact embryos can provide volumetric information about mutant phenotypes.
    MeSH term(s) Pregnancy ; Mice ; Animals ; Female ; Placenta ; Phenotype
    Language English
    Publishing date 2024-01-02
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.over107973
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    Kategorien

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  10. Article ; Online: Establishing Control Frequency of Embryonic and Fetal Loss in a Mutant Mouse Colony.

    Papaioannou, Virginia E / Behringer, Richard R

    Cold Spring Harbor protocols

    2024  Volume 2024, Issue 1, Page(s) pdb.prot108083

    Abstract: In the analysis of prenatal lethal recessive mutations, one must account for embryonic losses that are not related to the mutant phenotype. This protocol details the way to determine what the background level of unrelated embryonic loss is by a simple ... ...

    Abstract In the analysis of prenatal lethal recessive mutations, one must account for embryonic losses that are not related to the mutant phenotype. This protocol details the way to determine what the background level of unrelated embryonic loss is by a simple backcrossing strategy in the particular mouse strain that carries the lethal recessive mutation.
    MeSH term(s) Pregnancy ; Female ; Mice ; Animals ; Mutation ; Phenotype
    Language English
    Publishing date 2024-01-02
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.prot108083
    Database MEDical Literature Analysis and Retrieval System OnLINE

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