Cold Spring Harbor Protocols

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...

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...

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. For anyone planning to use mutational analysis for a research program, this article introduces an overall strategy for obtaining or generating a mutation and systematically analyzing the phenotype...

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.

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...

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.

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...

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.

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. From the isolation of clonal lines of targeted ES cells through the production of ES cell chimeras with the targeted ES cell clone, we discuss common technical problems and their most likely causes and solutions...

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...

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...

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...

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...

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...

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...

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...

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.

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.

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...

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...