Elsevier

Brain Research

Volume 835, Issue 1, 17 July 1999, Pages 18-26
Brain Research

Interactive report
Behavioral phenotyping of transgenic and knockout mice: experimental design and evaluation of general health, sensory functions, motor abilities, and specific behavioral tests1

https://doi.org/10.1016/S0006-8993(98)01258-XGet rights and content

Abstract

Rigorous experimental design can minimize the high risk of false positives and false negatives in the behavioral phenotyping of a new transgenic or knockout mouse. Use of well established, quantitative, reproducible behavioral tasks, appropriate Ns, correct statistical methods, consideration of background genes contributed by the parental strains, and attention to litter and gender issues, will maximize meaningful comparisons of −/−, +/−, and +/+ genotypes. Strategies developed and used by our laboratory are described in this review. Preliminary observations evaluate general health and neurological reflexes. Sensory abilities and motor functions are extensively quantitated. Specific tests include observations of home cage behaviors, body weight, body temperature, appearance of the fur and whiskers, righting reflex, acoustic startle, eye blink, pupil constriction, vibrissae reflex, pinna reflex, Digiscan open field locomotion, rotarod motor coordination, hanging wire, footprint pathway, visual cliff, auditory threshold, pain threshold, and olfactory acuity. Hypothesis testing then focuses on at least three well-validated tasks within each relevant behavioral domain. Specific tests for mice are described herein for the domains of learning and memory, feeding, nociception, and behaviors relevant to discrete symptoms of human anxiety, depression, schizophrenia, and drug addiction. An example of our approach is illustrated in the behavioral phenotyping of C/EBPδ knockout mice, which appear to be normal on general health, neurological reflexes, sensory and motor tasks, and the Morris water task, but show remarkably enhanced performance on contextual fear conditioning.

Introduction

Targeted gene mutation technology represents a powerful new tool for biomedical research. When the targeted gene is expressed in the brain, the behavioral phenotype of the mutant mice may reveal genetic mechanisms underlying normal behaviors, and may increase our knowledge of genetic factors in neuropsychiatric disorders. Transgenic mice have a new gene, or an additional copy of an existing gene, added to the genome. Knockout mice have a targeted gene deletion, such that no product of the mutated gene is synthesized in the null mutants. The methods for developing the mutation, and breeding strategies to generate null mutants in the F2 and subsequent generations, are extensively described in this volume and elsewhere 11, 54, 76.

Approximately 100 different genes expressed in the central nervous system have been targeted and phenotyped in transgenic and knockout mice to date 4, 11, 52, 54, 68. Reported behavioral phenotypes include aberrant social, reproductive, and parental behaviors, aggression, feeding disorders, learning and memory impairments, anxiety-like behaviors, and altered responses to antidepressants, antipsychotics, ethanol, and psychostimulant drugs of abuse. Experimental design is presently being optimized for thorough evaluation of behavioral phenotyping in mutant mice. This review is designed to suggest general methods that have been validated in our laboratory and others. Specific protocols for individual behavioral tasks can be found in the original publications referenced throughout the text, and in several recent reviews 11, 16, 19, 20, 34, 38, 54, 74.

Section snippets

The mice

After the mutation has been successfully introduced, and the gene product shown to be correctly overexpressed or absent, the first chimeras are mated with wildtype mice. A Mendelian distribution of F2 offspring is predicted. The strain of mouse used for the embryonic stem cells, for the donor blastulas, and for the breeding, can greatly affect the behavioral phenotype. For example, some 129 substrains (e.g. 129/J and 129/SvJ) have an incomplete or missing corpus callosum [42], and perform

Preliminary observations

A series of carefully conducted preliminary observations of general health, home cage behaviors, sensory abilities, and motor functions is first conducted for each mouse to avoid spurious false positives. If an animal has a major health problem or a gross motor defect it will be unable to perform many behavioral tasks for reasons not necessarily specific to the mutation. If an animal is blind or deaf, specific behavioral tests can be designed around the sensory deficit, such as olfactory

Specific behavioral tasks to test discrete hypotheses

Targeted gene mutation is designed to address specific hypotheses about the behavioral role of a gene. The set of specific behavioral tasks to be used for behavioral phenotyping is designed around the hypothesis. Genes for neuropeptides in the hypothalamus might be analyzed in tests of feeding behaviors, sexual behaviors, and stress-related behaviors. Signalling genes expressed in the hippocampus might be analyzed in tests of spatial learning and memory. Genes for serotonin receptor subtypes

Conclusions

In the opinion of this author, the present technology for the generation of mutant mice is most useful for modeling the symptoms of single gene mutation human hereditary diseases. Behavioral phenotypes identified for transgenic and knockout mice may lead to important medical applications. When the mutation is designed to mimic the genetic mutation in a human hereditary disease, the mouse behavioral phenotype can serve to evaluate the efficacy of new pharmacological and gene therapy treatments 4

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