Research reportAssessing the effects of the 129/Sv genetic background on swimming navigation learning in transgenic mutants: a study using mice with a modified β-amyloid precursor protein gene
Introduction
Gene targeting by homologous recombination in mouse embryonic stem (ES) cells has become an important tool for the analysis of genes thought to be involved in development and function of the central nervous system, in particular in learning and memory 16, 22, 35, 41. The Morris water maze [26]has been used to screen the learning capabilities of many mutants generated by gene targeting techniques. For a number of mutations it has revealed alterations of learning behavior 1, 2, 5, 7, 15, 19, 28, 30, 33, 46. In other studies, however, mutant mice failed to show significantly altered behavior in the water maze 4, 15, 25, 29. Reliable documentation and proper interpretation of the absence of negative behavioral effects may be as important as the analysis of deficits, for example for mutations with therapeutic or preventive potential. Such a case is the generation of scrapie resistant animals which lack a functional PrP gene 3, 43. Gene targeting in mice has also been used to determine whether normal expression of the amyloid precursor protein (βAPP) is essential for brain function 27, 47. This question is important, because the neuritic plaques which are a hallmark of Alzheimer's pathology are rich in amyloid β-peptide, a proteolysis product of βAPP. Thus, reduction of βAPP expression may provide ways to treat the disease [20].
The genetic background, i.e. the donor strain of the ES-cells used for the gene targeting procedure, is usually determined by technical considerations, such as availability of ES-cell lines, efficiency of breeding, and selection of transmitting animals by examination of fur color. ES-cells are often derived from inbred 129 (129/Sv, 129/J, 129/Ola) or BALB/c mice and transmitting chimeras are then mated to C57BL/6 mice [13]. Rapid results can be obtained if the mutation phenotype is investigated by comparing littermates in the F2 generation of such a cross. This type of procedure has also been employed in the recent generation and analysis of mice bearing a modification of the amyloid precursor protein (βAPP) gene which causes βAPP to be expressed at ca. 5% of wild-type levels and in a truncated form. We have found these mice to be severely impaired in learning the Morris water maze navigation task [27].
Pathology and survival rate of mice with induced mutations can change dramatically with different genetic backgrounds 9, 18, 32, 34, 36, indicating that the genetic background can profoundly affect the phenotype of a mutation. On the other hand, under baseline conditions, behavior and learning capabilities in the water maze show large differences between different inbred strains of mice 14, 17, 23, 38, 40, 42. In particular, the 129/Sv and BALB/c strains are known as poor swimming navigation learners 24, 44, while inbred C57BL/6 are fairly good learners 10, 31, 37, 38, 40. Moreover, the effect of anticholinergic treatment on water-maze learning in mice has been shown to depend on the genetic background 37, 39. However, in spite of much discussion 6, 8, 12, 13, 21, insufficient data is available documenting to which extent the genetic background can interfere with the behavioral characterization of mutants generated by gene targeting techniques. Here, we report that the effects of a modified βAPP gene on water-maze learning are masked in backcrosses to the 129/Sv(ev) strain.
Section snippets
Animals
The production of βAPP mutant mice has already been described in detail elsewhere [27]. In summary, a cassette containing a neomycin resistance gene and a transcription termination sequence was introduced into exon two of the βAPP gene of D3 ES cells, derived from 129/Sv(ev) mice (passage 13, obtained from E. Wagner). ES cells heterozygous for the interrupted βAPP gene were then injected into 3.5 day-old C57BL/6 blastocysts which were implanted into the uteri of pseudopregnant CD2 F1 mice. The
Comparison of parental strains and wild-type hybrid mice
The statistics of learning and retention effects within the experimental groups are shown in Table 1 and Table 2. The results of two-way ANOVA (genetic background by sex) and post-hoc Scheffé comparison of genetic backgrounds are summarized in Table 3 and Table 4. Group means of selected parameters are illustrated in Fig. 1. Three genetic backgrounds were compared: C57 (inbred C57BL/6), 129/Sv (inbred 129/Sv(ev) and wild-type 129-backcross), and hybrid (C57BL/6×129/Sv(ev), i.e. all wild-type
The APP mutation effect is confirmed
The results of swimming navigation testing of mutant and wild-type hybrid mice indicate that about half of the mutant mice had severely reduced escape performance and spatial orientation, while some mutant mice seemed less severely affected. Mutant mice seemed not principally unable to learn the task, but their acquisition was instable, retarded and inaccurate. Since the deficit in the affected mice prevented them from acquiring the task properly, they also showed poor scores for spatial
Acknowledgements
We thank C. Weissmann for continuous support and helpful discussions and F. Magara for critical reading of the manuscript. The study was supported by Swiss NF 31-42374.94 to D.P.W. and a Human Frontiers Science Program grant to H.P.L. We thank S. Sisodia for providing antibodies against APP.
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