Assessing 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

The Morris water maze is frequently used to screen mutant mice generated by gene targeting. Targeted ES-cells are often derived from 129/Sv or BALB/c mice, known as poor swimming navigation learners. After mating the founders with C57BL/6 mice, the F2 or F3 hybrid generation is typically used for behavioral testing. In hybrid 129/Sv×C57BL/6 mice, a modification of the βAPP gene entails impaired swimming navigation learning. This is readily detected despite behavioral variability, because wild-type 129/Sv×C57BL/6 hybrids outperform either of the parental strains and provide a control sample with good baseline performance. However, after backcrossing to the 129/Sv(ev) strain, the mutation effects are no longer detectable, masked by the very poor performance of wild-type 129/Sv(ev) mice. We conclude that F2 and F3 generations of 129/Sv×C57BL/6 crosses provide a suitable genetic background for behavioral testing of transgenic mice, provided that the samples are large enough to compensate for genetic and epigenetic variability and provided that normal performance in the control group is verified by comparison against a large database of mice tested under identical conditions. Creating congenic lines by backcrossing to an inbred strain is unlikely to enhance the sensitivity of the Morris test. Backcrossing to 129/Sv(ev) may even reduce it.     

Strain background influences neurotoxicity and behavioral abnormalities in mice expressing the tetracycline transactivator

The tet-off system has been widely used to create transgenic models of neurological disorders including Alzheimer's, Parkinson's, Huntington's, and prion disease. The utility of this system lies in the assumption that the tetracycline transactivator (TTA) acts as an inert control element and does not contribute to phenotypes under study. Here we report that neuronal expression of TTA can affect hippocampal cytoarchitecture and behavior in a strain-dependent manner. While studying neurodegeneration in two tet-off Alzheimer's disease models, we unexpectedly discovered neuronal loss within the dentate gyrus of single transgenic TTA controls. Granule neurons appeared most sensitive to TTA exposure during postnatal development, and doxycycline treatment during this period was neuroprotective. TTA-induced degeneration could be rescued by moving the transgene onto a congenic C57BL/6J background and recurred on reintroduction of either CBA or C3H/He backgrounds. Quantitative trait analysis of B6C3 F2 TTA mice identified a region on Chromosome 14 that contains a major modifier of the neurodegenerative phenotype. Although B6 mice were resistant to degeneration, they were not ideal for cognitive testing. F1 offspring of TTA C57BL/6J and 129X1/SvJ, FVB/NJ, or DBA/1J showed improved spatial learning, but TTA expression caused subtle differences in contextual fear conditioning on two of these backgrounds, indicating that strain and genotype can interact independently under different behavioral settings. All model systems have limitations that should be recognized and mitigated where possible; our findings stress the importance of mapping the effects caused by TTA alone when working with tet-off models.     

Speed congenics: applications for transgenic and knock-out mouse strains

Genetically modified mice are mainstream tools for medical research. The background strains used to generate transgenic and knockout mice have been studied extensively. In many instances, the background strain used to create the genetic modification is inappropriate for phenotypic analysis of the mutation. In such cases it is useful to develop a congenic strain of the transgenic mouse line in which the mutation is introduced into a more suitable genetic background. In this review, the approaches to generate congenic strains of transgenic and knockout mice will be discussed. In particular, marker-assisted selection protocols (MASP) will be discussed in detail to generate congenic strains in an accelerated fashion ("speed congenics").     

Behavioural analysis and susceptibility to CNS injury of four inbred strains of mice

Interpretation of data from gene targeting studies can be confounded by the inherent traits of the background inbred strains used in the generation of transgenic and null mutant mice. We have therefore compared the behaviour and response to CNS injury of four inbred strains commonly used in molecular genetic studies to produce models of neurological disease. Adult, male 129/Ola, BALB/c, C57BL/6 and FVB/N mice (2–4 months) were initially subjected to behavioural tests that comprised a neurological examination, determination of motor function and cognitive testing in the Morris water maze. Also the response to CNS injury following an acute kainic acid (KA) challenge (30 mg kg⁻¹, i.p.) was determined. The 129/Ola and BALB/c strains showed significant motor deficits when compared with the C57BL/6 and FVB/N strains. In contrast, only the FVB/N strain showed evidence of apparent cognitive impairments in the water maze as evidenced by increased pathlengths to locate the escape platforms and impaired performance in a probe trial. In addition, the FVB/N strain showed the most severe seizure response and mortality rate (62%) following administration of KA (30 mg kg⁻¹, i.p.). These behavioural changes were also associated with a greater degree of cell body and synaptophysin loss in the pyramidal CA3 hippocampal cell layer and astrogliosis 72-h post-dose. These data suggest that the FVB/N strain may not be the most suitable background strain for the development of new transgenic mice for the study of genes implicated in the learning and memory process.     

Central effects of a local inflammation in three commonly used mouse strains with a different anxious phenotype

As in humans, genetic background in rodents may influence a peculiar set of behavioural traits such as sensitivity to pain and stressors or anxiety-related behaviours. Therefore, we tested the hypothesis that mice with different genetic backgrounds [outbred (CD1), inbred (C57BL/6J) and hybrid (B6C3F1) adult male mice] display altered reactivity to pain, stress and anxiety related behaviours.We demonstrated that B6C3F1 mice displayed the more anxious phenotype with respect to C57BL/6J or CD1 animals, with the latter being the less anxious strain when tested in an open field and on an elevated plus maze. No difference was observed across strains in thermal sensitivity to a radiant heat source. Mice were then treated with a sub-plantar injection of the inflammatory agent Complete Freund's Adjuvant (CFA), 24 h later they were hyperalgesic with respect to saline exposed animals, irrespective of strain. We then measured intra-strain differences and CFA-induced inter-strain effects on the expression of various genes with a recognized role in pain and anxiety: BDNF, IL-6, IL-1β, IL-18 and NMDA receptor subunits in the mouse thalamus, hippocampus and hypothalamus. The more anxious phenotype observed in B6C3F1 hybrid mice displayed lower levels of BDNF mRNA in the hippocampus and hypothalamus when compared to outbred CD1 and C57BL/6J inbred mice. CFA led to a general decrease in central gene expression of the evaluated targets especially in CD1 mice, while BDNF hypothalamic downregulation stands out as a common effect of CFA in all three strains evaluated.     

Influence of genetic background and gender on bladder function in the mouse

Genetically targeted animals are used throughout research to investigate the role genes play in biological function, including the lower urinary tract. Generation of transgenic mice involves backcrossing for successive generations. Parental strain background genes can interact with the mutated gene potentially affecting interpretation of the mutant phenotype. Differences in physiological phenotypes may also be influenced by gender. The present study evaluated bladder function in five strains of male and female mice, 129S6/SvEvTac, A/J, B6129F1/Tac, BALB/cAnNCrL and C57BL/6NTacFBr. Urodynamic parameters were analyzed during infusion of saline (threshold and void volume, non-voiding contractions, pressure threshold and bladder contraction amplitude) in conscious mice and using voluntary urination in freely moving mice placed on filter paper (number of small and large diameter urine spots), which represent commonly used techniques in preclinical characterization of bladder function. Female BALB/c mice exhibited a significantly larger number of non-voiding contractions and urine dripping (increased number of small urine spots) compared to other female mice. Male BALB/c mice did not share this phenotype. Significant differences in threshold and void volumes were also noted amongst strains and genders. The numbers of large diameter urine spots differed amongst female, and not male, mouse strains. Gender differences were observed between sexes of the same strain in both large and small urine spots. These data demonstrate that genetic background and gender can influence bladder function in the mouse. These differences have a significant impact on the choice of strain and gender when investigating the effects of genetic manipulation on the micturition reflex.     

Impact of the genetic background of transgenic mice upon the formation and timing of choroid plexus papillomas

Transgenic mice harboring the SV40 large T antigen gene in a C57B1/6J genetic background (SV11) first express this gene at 1-2 weeks of age, develop papillomas of the choroid plexus by 80-90 days, and die within 125 days after birth. Transgenic mice harboring the same transgene in a (SV11-C57Bl/6J x NZW/lacJ) F1 genetic background express considerably lower levels of the transgene mRNA at comparable times after birth. As a consequence, tumor development and death are delayed. The NZW mice appear to contribute a dominant negative regulator for the expression of the SV40 large T antigen transgene, which in turn has a dramatic effect upon the time of appearance of tumors and the death of these transgenic animals.     

The relevance of inter- and intrastrain differences in mice and rats and their implications for models of seizures and epilepsy

It is becoming increasingly clear that the genetic background of mice and rats, even in inbred strains, can have a profound influence on measures of seizure susceptibility and epilepsy. These differences can be capitalized upon through genetic mapping studies to reveal genes important for seizures and epilepsy. However, strain background and particularly mixed genetic backgrounds of transgenic animals need careful consideration in both the selection of strains and in the interpretation of results and conclusions. For instance, mice with targeted deletions of genes involved in epilepsy can have profoundly disparate phenotypes depending on the background strain. In this review, we discuss findings related to how this genetic heterogeneity has and can be utilized in the epilepsy field to reveal novel insights into seizures and epilepsy. Moreover, we discuss how caution is needed in regards to rodent strain or even animal vendor choice, and how this can significantly influence seizure and epilepsy parameters in unexpected ways. This is particularly critical in decisions regarding the strain of choice used in generating mice with targeted deletions of genes. Finally, we discuss the role of environment (at vendor and/or laboratory) and epigenetic factors for inter- and intrastrain differences and how such differences can affect the expression of seizures and the animals' performance in behavioral tests that often accompany acute and chronic seizure testing.     

Survey of embryonic stem cell line source strains in the water maze reveals superior reversal learning of 129S6/SvEvTac mice

The availability of pluripotent embryonic stem (ES) cells for gene targeting has resulted in laboratory mice becoming important animal models of human neurological disease. Inbred strains of mice differ in many behavioural phenotypes, such that the same gene mutation can appear to have different phenotypic effects when introduced onto different genetic backgrounds. Prior knowledge of the behavioural phenotypes of the inbred strains used for gene targeting would, therefore, allow the selection of the most appropriate genetic background for the hypothesis to be tested. With this in mind, we tested eight strains of mice (129S1/SvImJ, 129S2/SvPasIcoCrlBR, 129S6/SvEvTac, B6129SF1/J, C57BL/6J, C57BL/6N, LP/J and SM/J), including the sources of five ES cell lines commonly used for gene targeting, in the spatial (submerged platform) version of the Morris water maze, the most widely used paradigm to evaluate the cognitive abilities of genetically modified mice. The three 129 substrain sources of ES cell lines demonstrated spatial learning in the water maze that was superior to that of C57BL/6J, the inbred strain most commonly used for the maintenance and phenotypic testing of mutations. In addition, 129S6/SvEvTac was unique amongst the eight strains tested in having a particular capacity for reversal learning, when the submerged platform was relocated to the opposite quadrant. We conclude that some substrains of 129 could provide suitable genetic backgrounds for testing gene mutations that might be expected to impair cognitive function, thus negating the need to backcross to C57BL/6J, thereby avoiding the so-called "flanking gene problem".     

Background and gender effects on survival in the TgN(SOD1-G93A)1Gur mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder. While most cases of ALS are sporadic, 10-15% are familial, and of these 15-20% possess a mutation in the gene that codes for the enzyme Cu/Zn superoxide dismutase (SOD1). In families of ALS patients with specific SOD1 mutations, affected members demonstrate significant heterogeneity of disease and a large variation in age of onset and severity, suggesting that there are genetic modifiers of disease expression. Transgenic mice expressing mutant forms of SOD1 demonstrate symptoms similar to those seen in patients with ALS. We have observed in our colony of G93A SOD1 transgenic mice a milder phenotype in mice in a C57BL/6J background than the C57BL/6JxSJL/J hybrid background used by Jackson Laboratories to maintain their colony. To investigate the effect of genetic background on phenotype, we have constructed congenic lines on two genetic backgrounds, C57BL/6J (B6) and SJL/J (SJL). We report the influence of background and gender on the survival of these congenic lines compared to the hybrid C57BL/6JxSJL/J background. The mean survival of G93A SOD1 mice in the hybrid B6/SJL background was 130 days, with females surviving significantly longer than males. When compared to the hybrid B6/SJL background, the survival of mice in the SJL background significantly decreased, and the gender difference in survival was maintained. On the other hand, mean survival in the B6 background significantly increased, and in contrast to the B6/SJL and SJL backgrounds, there was no difference in survival between males and females. Transgene copy numbers were verified in all animals to ensure that any phenotypic differences observed were not due to alterations in copy number. This is the first report of a shortened lifespan when the G93A SOD1 transgene is placed on the SJL/J background and an increased survival with the loss of gender influences when the transgene is placed on the C57BL/6J background.     

Phenotypic expression of the targeted null-mutation in the dopamine transporter gene varies as a function of the genetic background

The dopamine transporter (DAT) plays a critical role in calibrating the duration and intensity of dopamine (DA) neurotransmission. Mice in which the DAT gene has been genetically deleted exhibit constitutively high levels of extrasynaptic DA and spontaneous hyperactivity. Numerous studies have characterized the adaptive molecular, physiological, and behavioural consequences of abnormal DA neurotransmission in these mice. In order to determine the genetic background contribution to these phenotypes, the DAT mutation was transferred on C57BL/6JOrl (B6) or DBA/2JOrl (D2) inbred backgrounds for more than ten generations of back-crossing to derive three B6-, D2-, and B6xD2(F(1))-DAT strains. We observed that the genetic background dramatically affects phenotypes previously reported on DAT knockout (KO) mice. Depending on the genetic background, it was possible to restore survival, growth rate and ability to lactate. Interactions with the genetic background were found to modulate both quantitative and qualitative patterns of novelty-driven spontaneous hyperactivity. The paradoxical calming effect of cocaine was observed for all DAT-KO mice. However, the genetic background influenced individual threshold responses to both locomotor and rewarding effects of cocaine. These findings reveal the extent of phenotypic variation associated with the DAT mutation. They also provide concrete arguments against the assumption that the normal function of a gene can be inferred directly from its mutant phenotype.     

Performance of F2 B6x129 hybrid mice in the Morris water maze, latent inhibition and prepulse inhibition paradigms: Comparison with C57Bl/6J and 129sv inbred mice

Assessment of cognition and information processing in mice is an important tool in preclinical research that focuses on the development of cognitive enhancing drugs. Analysis of transgenic (TG) and knockout (KO) mice is usually performed on a F2 B6x 129 background. In the present study, we have compared performance of F2 B6x 129 hybrid mice (F2 mice) with that of the two parental inbred strains (C57Bl/6J and 129sv mice), and a wild-type (WT) strain (with a combined B6x 129 background) in three cognitive/information processing paradigms. It was found that the F2 mice outperformed either of the parental strains and provide a control sample with good baseline performance in the Morris water maze (MWM). Reliable deficits could be obtained in learning and memory in this paradigm following injections with scopolamine (0.16 mg/kg) in the F2 mice, which can potentially be used to test effects of reference and novel compounds in order to develop cognitive enhancing drugs. Furthermore, it was shown that the four genotypes showed normal latent inhibition (LI) using the conditioned taste aversion (CTA) paradigm and exhibited no differences in prepulse inhibition (PPI) levels. Following the setup of these procedures in mice, we are now able to compare the effects of gene knockout/mutations used for target validation with results in the present study as a frame of reference.     

A behavioural characterisation of the FVB/N mouse strain

The use of transgenic models in scientific research has made an enormous contribution to our understanding of the causes and symptoms of many diseases, including neurodegenerative conditions such as Alzheimer's Disease (AD) and Parkinson's Disease (PD). In the creation of transgenic models of neurodegenerative disease, effects of the background strain of the animal on the resulting genotype must be taken into consideration. This is particularly true for behavioural studies in which the background strain of the mouse may mask the phenotype of the genetic manipulation. Here, the behaviour of two mouse strains used in transgenic models, FVB/N and C57BL6/J, were compared. Studies of circadian wheel activity, cognition and aggression revealed considerable phenotypic differences between strains. These data also indicate that the FVB/N strain is not appropriate as a background strain in the behavioural assessment of transgenic mouse models.     

Strain-specific regulation of striatal phenotype in Drd2-eGFP BAC transgenic mice

Mice carrying bacterial artificial chromosome (BAC) transgenes have become important tools for neuroscientists, providing a powerful means of dissecting complex neural circuits in the brain. Recently, it was reported that one popular line of these mice--mice possessing a BAC transgene with a D(2) dopamine receptor (Drd2) promoter construct coupled to an enhanced green fluorescent protein (eGFP) reporter--had abnormal striatal gene expression, physiology, and motor behavior. Unlike most of the work using BAC mice, this interesting study relied upon mice backcrossed on the outbred Swiss Webster (SW) strain that were homozygous for the Drd2-eGFP BAC transgene. The experiments reported here were conducted to determine whether mouse strain or zygosity was a factor in the reported abnormalities. As reported, SW mice were very sensitive to transgene expression. However, in more commonly used inbred strains of mice (C57BL/6, FVB/N) that were hemizygous for the transgene, the Drd2-eGFP BAC transgene did not alter striatal gene expression, physiology, or motor behavior. Thus, the use of inbred strains of mice that are hemizygous for the Drd2 BAC transgene provides a reliable tool for studying basal ganglia function.     

Progressive CAG expansion in the brain of a novel R6/1-89Q mouse model of Huntington's disease with delayed phenotypic onset

Transgenic models representing Huntington's disease (HD) have proved useful for understanding the cascade of molecular events leading to the disease. We report an initial characterisation of a novel transgenic mouse model derived from a spontaneous truncation event within the R6/1 transgene. The transgene is widely expressed, carries 89 CAG repeats and the animals exhibit a significantly milder neurological phenotype with delayed onset compared to R6/1. Moreover, we report evidence of progressive somatic CAG expansions in the brain starting at an early age before an overt phenotype has developed. This novel line shares a common genetic ancestry with R6/1, differing only in CAG repeat number, and therefore, provides an additional tool with which to examine early molecular and neurophysiological changes in HD.     

The relevance of individual genetic background and its role in animal models of epilepsy

Growing evidence has indicated that genetic factors contribute to the etiology of seizure disorders. Most epilepsies are multifactorial, involving a combination of additive and epistatic genetic variables. However, the genetic factors underlying epilepsy have remained unclear, partially due to epilepsy being a clinically and genetically heterogeneous syndrome. Similar to the human situation, genetic background also plays an important role in modulating both seizure susceptibility and its neuropathological consequences in animal models of epilepsy, which has too often been ignored or not been paid enough attention to in published studies. Genetic homogeneity within inbred strains and their general amenability to genetic manipulation have made them an ideal resource for dissecting the physiological function(s) of individual genes. However, the inbreeding that makes inbred mice so useful also results in genetic divergence between them. This genetic divergence is often unaccounted for but may be a confounding factor when comparing studies that have utilized distinct inbred strains. The purpose of this review is to discuss the effects of genetic background strain on epilepsy phenotypes of mice, to remind researchers that the background genetics of a knockout strain can have a profound influence on any observed phenotype, and outline the means by which to overcome potential genetic background effects in experimental models of epilepsy.     

Fierce: a new mouse deletion of Nr2e1; violent behaviour and ocular abnormalities are background-dependent

A new spontaneous mouse mutation named fierce (frc) is deleted for the nuclear receptor Nr2e1 gene (also known as Tlx, mouse homolog of Drosophila tailless). The fierce mutation is genetically and phenotypically similar to Nr2e1 targeted mutations previously studied on segregating genetic backgrounds. However, we have characterized the fierce brain, eye, and behavioural phenotypes on three defined genetic backgrounds (C57BL/6J, 129P3/JEms, and B6129F1). The data revealed many novel and background-dependent phenotypic characteristics. Whereas abnormalities in brain development, hypoplasia of cerebrum and olfactory lobes, were consistent on all three backgrounds, our novel finding of enlarged ventricles in 100% and overt hydrocephalus in up to 30% of fierce mice were unique to the C57BL/6J background. Developmental eye abnormalities were also background-dependent with B6129F1-frc mice having less severe thinning of optic layers and less affected electroretinogram responses. Impaired regression of hyaloid vessels was observed in all backgrounds. Furthermore, retinal vessels were deficient in size and number in 129P3/JEms-frc and B6129F1-frc mice but almost entirely absent in C57BL/6J-frc mice. We present the first standardized behavioural tests conducted on Nr2e1 mutant mice and show that C57BL/6J-frc and B6129F1-frc mice have deficits in sensorimotor assays and are hyperaggressive in both sexes and backgrounds. However, C57BL/6J-frc mice were significantly more aggressive than B6129F1-frc mice. Overall, this extensive characterization of the fierce mutation is essential to its application for the study of behavioural, and brain and eye developmental disorders. In addition, the background-dependent differences revealed will enable the identification of important genetic modifiers.     

Differences in nociceptive behavioral performance between C57BL/6J, 129S6/SvEv, B6 129 F1 and NMRI mice

The interpretation of knockout and transgenic mouse studies in pain research critically depends on detailed knowledge of the performance profile of the background strains. Pain-related behavior was compared between four relevant mouse strains (C57BL/6J, 129S6/SvEv, B6 129 F1 and NMRI mice of both sexes) using an extended test battery that included an unusual variety of assays for thermal and mechanical acute nociception, and inflammatory and neuropathic pain. Strain- and gender-dependent differences were demonstrated in many of these nociceptive assays. Particularly, C57BL and 129 mice, which serve as the default genetic backgrounds for experiments in genetically altered mice, display quite different patterns of nociceptive performance. Compared to C57BL/6J mice, 129S6/SvEv animals are less sensitive to inflammatory pain conditions (thermal sensitivity after carrageenan subplantar injection; flinch behavior after formalin injection), while the opposite is observed in the neuropathic pain condition and the visceral pain model. These data may be of special interest for genetic studies, where issues related to the background phenotype may confound their interpretation.