Genetic background. Most commonly used ES cell lines are derived from several129 substrains. Chimeric animals are typically bred to C57BL/6 mice, producing genetically similar F1 animals sharing similar chromosomal complements of 129 and C57BL/6 strains. However, when these animals are crossed to generate F2 animals homozygous for the target locus, the offspring have a random mix of 129 and C57BL/6 chromosomal DNA. Ideally, the genetic background of control and experimental animals should be identical, with exception of the target locus. In cases where background effects are likely to be important, the target locus is best propagated in congenic strains by successive backcrossing to one inbred strain. After breeding parental strains, F1 progeny are bred back to one parental strain (usually C57BL/6). F2 progeny from this mating are then similarly bred back to the parental strain until, after 6 backcross breedings (a process which generally takes two years), the resultant offspring are 99% similar to the chosen strain, with the exception of the region surrounding the target locus. However, circumstances frequently dictate that phenotype of genetically altered animals be evaluated long before congenic strains can be generated. In these case, wild-type and heterozygous littermates from the same breedings are used as controls. To ensure that the range of phenotypes possible due to genetic recombination is assessed, large numbers of animals should be examined.
Compensation. When a gene of interest is disrupted, a compensatory process may take over the function of the missing gene and mask its phenotypic outcome. Such compensation is typically identified by the altered or increased expression of related genes in the presence of a relatively normal phenotype.
Epigenetic phenomena. Environmental phenomena such as stress and food composition can have substantial effects on phenotype. In particular, behavioral phenotypes, or those phenotypes such as obesity which are affected by behavior and feeding can be particularly affected.
Infectious disease status. If animals have been produced at the research facility, information regarding the health status of the room in which they live will be available. In general, those phenotypes which affect the immune system are most likely to suffer potential confounding effects of a prevalent but subclinical infectious disease. Not infrequently, Helicobacteriosis will present as clinical disease (rectal prolapse), particularly in animals prone to inflammatory bowel disease as the result of ablation of components of their immune system.
Strain-specific or age-related background pathology. Online resources such as the Mouse Phenome Database, the database of Inbred Strain Characteristics, and the Mouse Tumor Biology Database provide searchable databases of strain-specific anatomy and pathology (see Information Resources for Phenotyping: Pathology and Morphology).
Two issues bear special consideration when selecting animals for morphologic evaluation - the age at which animals are evaluated and their genetic background. The window during which the phenotype is expressed will determine the optimal age for comprehensive examination. For progressive conditions, animals in early, mid and late stages of the condition should be chosen for histologic analysis. Not uncommonly, no clinical abnormalities are noted at all. In this case, baseline phenotyping can be performed in young (8-12 weeks) and older (12-15 months) adult animals.
The effects of genetic background on phenotype are well documented, and can be substantive enough to obscure subtle phenotypes originating from the induced genetic alteration. Ideal candidates for phenotypic evaluation would be backcrossed -/-, +/- and +/+ littermates from at least the 5th generation. These animals would be approximately 95% genetically similar, and would differ only in the region surrounding the target gene. In practice, the constraints of time, cost and potential loss of the phenotype from multiple backcrosses result in presentation of animals of more mixed background for examination. This does not pose particular problems if the phenotype attributable to the target gene is an obvious one. However, the more subtle the phenotype, the greater the number of animals that are needed to extract a significant result from the experiment. At a minimum, two age matched, sex-matched individuals of each genotype (at each time point, and in the case of transgenic animals, from each founder line) are needed to obtain baseline histologic data.
- Head - nasal passages, teeth, skin, vomeronasal organ, eyes, inner, middle and outer ear, tongue, salivary glands, brain, pituitary gland, bone.
- Heart (longitudinal section), lung (whole lung), trachea and thyroid glands.
- Liver, gall bladder, adrenal glands and kidneys (in transverse and longitudinal section).
- Stomach, duodenum, jejunum, pancreas, mesenteric lymph nodes.
- Ileum, cecum and colon, rectum.
- Urinary bladder, testes, accessory sex glands, preputial gland (in males) or uterus, ovaries, clitoral gland and mammary gland (in females).
- Cervical lymphoglandular complex (cervical lymph node, submandibular, submaxillary and parotid lymph nodes), diaphragm.
- Hind and forelimbs and sternum (skeletal muscle, bone marrow, peripheral nerve, skin, joints)
Collection of embryos at specific developmental stages. Matings between fertile males and spontaneously cycling females are usually set up in the late afternoon or early evening. Females in proestrus can be selected by vaginal inspection. Approximately half of the females selected this way will mate that night. Consequently, a relatively large number of matings need to be set up in order to obtain the required number of timed pregnant females. Observation of a vaginal plug is required to accurately determine the developmental stage of embryos. In mice kept in a standard 12 hour light:dark cycle, it is assumed that mating occurs at the mid-dark point, at approximately 2 am. If a vaginal plug is identified the next morning, embryos will be assumed to be E 0.5 (embryonic day) or 0.5 dpc (days post coitum) old. Implantation usually occurs at E4.5 and the duration of pregnancy is 19.5-21 days. Before implantation, embryos may be retrieved by flushing the oviduct and uterus with phosphate buffered saline. Between E4.5 and E8.5, it is best to isolate the embryo within its intact decidual swelling to avoid damaging it. After E8.5, the embryo can be dissected from the uterus and its yolk sac. It can be retained within the amnion, but considerable care should be taken to avoid damage
Fixation, embedding and orientation. Embryos may be fixed in Bouin's solution, 10% formalin or 4% paraformaldehyde. Particularly with Bouin's solution, the tissue will become brittle if placed in fixative for too long. Embryos with a crown-rump length of 2mm require only 1 hour in fixative, while those with a crown-rump length of approximately 15 mm can be placed in fixative for up to 24 hours. After removal from fixative, embryos may be placed in 70% ethanol for long-term storage at room temperature.