Disease susceptibility implications of preferential inactivation of the paternal X chromosome in extraembryonic endoderm of the mouse.

In the mouse, there is preferential inactivation of the paternally-derived X chromosome in extraembryonic tissues of early embryos, including trophectoderm and primitive endoderm or hypoblast. Although derivatives of these tissue have long been considered to be purely extraembryonic in nature, recent studies have shown that hypoblast-derived cells of the 'extraembryonic' visceral endoderm make a substantial cellular contribution to the definitive gut of the fetus. This raises questions about the eventual fate of these cells in the adult and potential disease implications due to the skewed inactivation of the paternally derived X in females heterozygous for X-linked mutations.

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.

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.

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.

Phenotypic Analysis of Preimplantation Mouse Embryos in Culture.

Preimplantation embryo culture is a valuable approach to investigate a preimplantation lethal phenotype. Standard culture methods have been perfected such that the entire preimplantation period and the process of implantation can be followed in vitro. This protocol provides modifications for the analysis of clutches of embryos from heterozygous matings specifically for the purpose of distinguishing a preimplantation phenotype in homozygous mutants.

Orientation of Implanted or Dissected Mouse Embryos for Histological Sectioning.

Histological analysis is an informative part of any mutational analysis. Interpretation of histological sections is much easier, and comparisons of mutant and wild-type embryos are more reliable if the plane of sectioning is precisely controlled. This protocol provides methods for obtaining sections with reproducible orientation for embryos between E4.

Phenotypic Analysis of Preimplantation Lethality in Mice.

Preimplantation development covers a period of ∼4.5 d from fertilization to implantation in the uterus. If a homozygous mutant phenotype causes the death of embryos during this period, simple culture methods are available that support preimplantation development to allow a thorough morphological assessment.

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.

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.

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.

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.

Strategies for the Production and Phenotypic Analysis of Mutations in the Mouse.

The laboratory mouse is one of the primary model organisms for human biology and genetics. The production and phenotypic analysis of mutations in the mouse has made great strides with targeted mutagenesis in embryonic stem (ES) cells and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene editing in preimplantation embryos. Mutations in many genes are now available or can be easily produced.

Cross-Fostering of Newborn Mice to Counteract Bad Parenting.

Newborn mice can be cross-fostered to a lactating female if the birth mother is not taking care of them properly. The pups are removed from the birth mother, warmed, and placed in a clean cage with a recently postpartum, lactating female. Hints on improving the chances of successful fostering are provided.

Phenotypic Analysis of Perinatal Lethality in Mice.

If homozygous mutants are found dead shortly after birth, further analysis of the phenotype will depend on knowing whether death occurred before or after parturition, which marks a major shift in physiological conditions of neonates. This overview provides methods for determining the time of death and discusses possible causes of lethality during this period, including catastrophic morphological abnormalities or developmental delay of some or all organs. Attention is given to specific defects that could result in perinatal death, such as cranial nerve defects, cleft palate, diaphragm defects, and other problems that might not have been relevant during intrauterine development but become critical during the transition to extrauterine existence.

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.

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.

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.

Embryonic Stem Cell Gene Targeting and Chimera Production in Mice.

Producing a custom gene mutation in embryonic stem (ES) cells, whether through homologous recombination or CRISPR-Cas gene editing, is the first step along the way to getting the mutation into live mice. However, there are a number of additional steps along the way, each presenting technical challenges. Here, we provide a guide for troubleshooting when the results are not as expected and to distinguish technical problems from possible biological effects of the mutation.

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.

Tissue- and/or Temporal-Specific Mutations in Mice Using Conditional Alleles.

Although many existing mutations are null alleles, multipurpose conditional alleles that can be used to delete gene function in a tissue- and/or temporal-specific manner are increasingly the alleles of choice. There are two distinct but related advantages: first, early lethal effects of the mutation can be bypassed by leaving the gene intact until later stages in development; second, indirect or secondary effects on an organ of interest can be eliminated by tissue- or organ-specific gene deletion. In this overview, we cover aspects of testing and using conditional alleles to ensure that the desired effect is obtained, including how to test the engineered conditional allele to ensure it functions as planned, and how to test any recombinase mouse strain used, including inducible transgenic or knock-in lines.

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.

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.

Counting Murine Corpora Lutea to Determine the Number of Oocytes Ovulated.

One can determine if and how many oocytes have been ovulated in a female mouse by counting the number of corpora lutea (CL) on the ovaries during the process of preimplantation embryo collection. A simple method of harvesting the ovaries and observing with a dissecting microscope and top lighting is provided along with a description of how to recognize CL. As the embryos rarely, if ever, cross the uterotubal junction, this provides a measure of the maximum number of embryos expected to be recovered from each oviduct or uterine horn, a number that can be valuable in analyzing early lethal mutations.

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.