Mouse embryos, chimeras, and embryonal carcinoma stem cells-Reflections on the winding road to gene manipulation.

The relationship of embryonal carcinoma (EC) cells, the stem cells of germ cell- or embryo-derived teratocarcinoma tumors, to early embryonic cells came under intense scrutiny in the early 1970s when mouse chimeras were produced between EC cells and embryos. These chimeras raised tantalizing possibilities and high hopes for different areas of research. The normalization of EC cells by the embryo lent validity to their use as in vitro models for embryogenesis and indicated that they might reveal information about the relationship between malignancy and differentiation. Chimeras also showed the way for the potential introduction of genes, selected in EC cells in vitro, into the germ line of mice. Although EC cells provided material for the elucidation of early embryonic events and stimulated many studies of early molecular differentiation, after years of intense scrutiny, they fell short as the means of genetic manipulation of the germ line, although arguably they pointed the way to the development of embryonic stem (ES) cells that eventually fulfilled this goal.

Sex Genotyping Mice by Polymerase Chain Reaction.

A simple method to determine the genetic sex of a mouse is to amplify DNA from a male-specific gene by polymerase chain reaction (PCR). This protocol is used to detect the Y-chromosome-specific gene Sry in tissue lysates of tail tip or ear punch samples.

Establishing Control Frequency of Embryonic and Fetal Loss in a Mutant Mouse Colony.

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.

Special Breeding Techniques for Use in Mouse Mutation Analysis.

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.

Analysis of Mid- to Late-Gestation Phenotypes in Mice.

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.

Getting around an Early Lethal Phenotype in Mice with Chimeras.

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.

Uncovering Phenotypes in Mutant Mice by Determining Embryo, Organ, Tissue, and Cell Developmental Potential.

The death of an embryo during gestation does not necessarily preclude the study of the mutant embryo or the developmental potential of its individual cells, tissues, or organs. Whole-embryo in vitro culture prior to the time of death will allow real-time observation of living embryos and direct comparisons with controls. Organ anlage can be removed from embryos and cultured in vitro beyond the time of death of the whole embryo. In both whole embryos and organ anlage culture, fluorescent protein reporters may be used productively to follow cell types or specific gene expression changes. Some cells, such as hematopoietic cells, and organ anlage, may be suitable for transplantation to wild-type hosts for further analysis of their potential. Additionally, cell lines, including embryonic stem (ES) cells, trophoblast stem (TS) cells, extraembryonic endoderm (XEN) stem cells, and epiblast-derived stem cells (EpiSC), can be derived from mutant embryos to reveal the potential of the mutant cells outside the context of the whole organism. Mutant stem cells or even whole mutant embryos can be used to test potential in chimeras or in teratomas.

Strategies for Maintaining Mouse Mutations.

Rules for naming a new mutation are provided. The majority of new mutations are recessive and thus easily maintained in a mouse strain. Considerations on the choice of genetic background are given, depending on how the mutant was produced and how you intend to analyze it. General information on maintaining a mutant colony to perpetuate the mutation and to efficiently produce homozygous mutant mice for analysis is provided. Also discussed are special breeding techniques to delete a selection cassette in vivo, if you produced the mutation in embryonic stem (ES) cells, and to maintain a mutant with a balancer chromosome. In the event of either male or female infertility in the heterozygotes, assisted reproductive techniques may be necessary.

Necropsy Guide for the Collection of Tissues from Mice with or without Tumors.

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.

Phenotypic Analysis of Dominant Mutant Effects in Mice.

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.

Isolation of XO Subclones from XY Murine Embryonic Stem Cells.

This is a simple procedure to isolate XO subclones from XY murine embryonic stem cells in situations that require transmission of a mutation through the female germline-for example, if the mutation adversely affects spermatogenesis. XY cells are plated at clonal density, and resulting colonies are genotyped by polymerase chain reaction for a Y-specific probe to identify clones that have spontaneously lost the Y chromosome.

Recovering a Targeted Mutation in Mice from Embryonic Stem Cell Chimeras or CRISPR-Cas Founders.

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.

Phenotypic Analysis: Assessing Timing of Recessive Prenatal Lethality in Mice.

Once a recessive mutation has been established in a mouse strain in the heterozygous state, the task of phenotypic analysis of the homozygous mutants can begin. This overview leads you through a sequence of steps to determine whether the homozygous mutants are present at birth or whether the mutation causes prenatal lethality. In the case of a prenatal lethality, the time of death of the mutants, which could occur at any time during pre- or postimplanation development, must be firmly established before further phenotypic analysis. Here, we present a detailed plan to efficiently determine the time of prenatal death of the mutants and provide a guide for developmental landmarks to establish how far they progress during gestation. To determine whether or not homozygous mutants are present or normal at any given time point, it is important to recover a sufficient number of embryos. Examples of a simple Chi square test for Mendelian segregation is provided to establish statistical significance for the genotype/phenotype distribution.

Analysis of Postnatal Mutant Phenotypes in Mice.

Viable homozygous mutant newborn mice may show effects of a mutation at any time during their development by exhibiting abnormal structure, function, or lethality. This overview guides the analysis of postnatal mice through gross anatomical assessment and the detection of visible phenotypes prior to weaning such as altered growth patterns, neurological problems, or abnormalities in movement or coordination. Advice on marking pups for identification purposes and providing adequate nutrition in the event of eating problems is given. After weaning and at the onset of puberty, different phenotypes may become manifest, including compromised growth and vigor and reproductive problems in males and/or females. Assessing infertility in each sex is addressed.

The "No Phenotype" Challenge in Analyzing Mutant Mice.

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.

Backcrossing to Generate a Congenic Mouse Strain.

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.

Mouse Gene-Targeting Strategies for Maximum Ease and Versatility.

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.

Cell Counting Techniques for Preimplantation Mouse Embryos Using Fluorescent DNA Dyes.

Counting cells in preimplantation embryos by light microscopy is straightforward until morula compaction, when cell boundaries in living embryos become indistinct. An alternative to morphological assessment of cell number is to use fluorescent DNA dyes. This protocol details simple nuclear counting with a single DNA dye (Hoechst) or a more complicated procedure in which differential nuclear counts of the trophectoderm and inner cell mass (ICM) can be made using immunosurgery of the blastocyst and two DNA dyes (Hoechst and propidium iodide).

Phenotypic Analysis of Periimplantation to Mid-Gestation Lethality in Mice.

Periimplantation to mid-gestation lethality is indicated if no living homozygous mutants are recovered at E12.5 and the number of empty implantation sites or degenerating/abnormal embryos fits the expected number of homozygous mutants. To determine the time of death, this overview details the characteristic features of lethality shortly after implantation (E4.5-E5.5) or lethality between gastrulation and allantoic fusion (E6.5-E9.5). Determining the phenotype of the mutants involves making a gross morphological assessment, staging the embryos, and photodocumenting any abnormalities. Further levels of analysis discussed are histological assessment, molecular characterization of gene expression in the mutant embryos, and measurements of cell proliferation and cell death.

Obtaining or Generating Gene Mutations in Mice.

The starting point in a mutational analysis of gene function is obtaining or producing a mutant. Here different methods of obtaining mouse mutants are discussed, including screening for spontaneous mutants, screening for mutants following chemical or X-ray mutagenesis, and producing mutations through targeted manipulation of the genome. Manipulation of the genome can be random, as in different types of insertional mutagenesis. Alternatively, targeted manipulation such as gene targeting using homologous recombination in embryonic stem (ES) cells or gene editing by CRISPR-Cas can be used to produce custom mutations in a specific gene. The basic methods are outlined, and the advantages and disadvantages of homologous recombination and CRISPR-Cas gene editing are discussed. Resources for obtaining mutations that already exist are provided. If, for your planned study, no suitable mutations are available, there is advice about what you should know about your gene of interest before embarking on a gene targeting experiment.