Methodology Aspects of Colony Maintain for a Murine Model of Amyotrophic Lateral Sclerosis (ALS) TDP-43 Proteinopathy

Simple Summary Rodent models of central nervous system (CNS) disorders are widely used to explore pathology and molecular mechanisms of disease. These models are a valuable tool for understanding the genetic basis of amyotrophic lateral sclerosis (ALS) research, a CNS disease that affects motor neurons in the brain and spinal cord, causing loss of voluntary muscle control. There are two different types of ALS: Sporadic (sALS), accounts for 90 to 95 percent of all cases, which means the disease seems to occur at random with no clearly associated risk factors and no family history of the disease, and familial (fALS), which is inherited and accounts for 5 to 10 percent of all cases. One example of a gene mutation in fALS is the A315T mutation to the gene encoding TDP-43. The congenic Prp-TDP43A315T Tg model of ALS disease expresses full-length human TARDBP containing this mutation. The effects of the highly aggressive phenotype of this Tg model of human ALS, and its premature sudden death prior to full development of neurodegenerative symptoms, makes it essential to determine their reproductive pattern in order to guarantee proper colony maintenance, which is the main goal of this study. Abstract The use of genetically engineered mouse (GEMs) models provides an unprecedented opportunity to study the genetic basis of diseases and gene function, therefore it is paramount to determine reproductive parameters that guarantee proper colony maintenance. We studied the reproductive parameters of mice hemizygous for TDP-43A315T transgene, which are viable, fertile, and express a mutant human TAR DNA binding protein (hTDP-43) cDNA harboring an amino acid substitution associated with familial amyotrophic lateral sclerosis (fALS). TDP43A315T mice were backcrossed to a C57Bl6/J pure background for four consecutive generations. The Tg offspring genotype were then confirmed by PCR assays. Our statistical analysis indicated there were no differences in the sex and number of pups per offspring when hemizygous female and male TDP43A315T mice were backcrossed to C57Bl6/J mice. Interestingly, our results showed significant differences in the number of offspring expressing the transgene when hemizygous TDP43A315T male mice were used as breeders. Therefore, our findings suggest that male TDP43A315T mice transfer the transgene with a greater genetic strengths. Such is an important breeding consideration to ensure the principle of reduction in animal experimentation considering most basic research with models focuses on males and excludes female mice.


Introduction
Genetically engineered mouse (GEMs) models are essential to understand the fundamental mechanisms underlying the onset of malignance, and to discover improved methods to prevent, diagnose, and treat a wide spectrum of diseases [1]. More specifically, GEMs models are valuable tools for the genetic understanding of amyotrophic lateral sclerosis (ALS) research [2]. ALS is a motor neuron disease (MND) characterized by the selective and progressive loss of upper and lower motor neurons of the cerebral cortex, brainstem, and the spinal cord [3]. The majority of patients suffer from sporadic ALS (sALS; >90%), while only a small subset of patients suffers from inherited ALS (fALS; <10%) 20% of cases have been linked to mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) [4], which represent the 2% of all cases of fALS. In 1993, Rosen and collaborators discover SOD1 mutations [5], which then led to the development of numerous models of Tg SOD-1 mice that simulate an ALS-like disease. However, the recent discovery of other highly relevant genetic disease-mutations such as TAR DNA binding protein (TDP-43) [6,7], bring new powerful GEMs models to the researchers in the field to explore the role this gene mutation plays on this complex neurological disorder.
Prp-TDP43 A315T Tg model of ALS disease containing the A315T mutation is seen in fALS patients [8], under the control of the mouse prion protein (PrP) promoter [9,10]. In terms of ALS pathology, these mice develop neuropathology and behavioural deficits similar to ALS and frontotemporal dementia (FTD), including cytoplasmic TDP-43 inclusions, neuroinflammation, axonal pathology, and cognitive and motor impairment. However, they do not develop neuronal loss or paralysis [9]. The initial report published by Wegorzewska et al. [9] indicated that Prp-TDP43 A315T mice were born at normal inheritance ratios, weighed the same as non-Tg littermates, and appeared normal up to three months of age. In this study they demonstrated that Tg Prp-TDP43 A315T mice have an average survival of approximately 154 ± 19 days. However, hemizygous male mice for TDP-43 A315T transgene die almost 1 month earlier than females [9]. Indeed, the lifespan of male hemizygous for TDP-43 A315T transgene appeared to be reduced when these mice were crossed with C57BL6/J pure background [11]. Coughlan et al. [12] and other research groups [11,[13][14][15] determined a premature sudden death prior to full development of neurodegenerative symptoms. However, in 2014 Herdewyn et al. [16] demonstrated that the range of survival of Prp-TDP43 A315T mice can be extended using special diets to mitigate the intestinal dysmotility phenotype and sudden death. What is more, the results also highlighted the survival differences between Tg Prp-TDP43 A315T mice in females and males were still present [16]. As reported, these studies indicate Tg Prp-TDP43 A315T female mice have a much longer breeding window than male mice hemizygous for TDP-43 A315T transgene.
Therefore, determining the reproductive parameters of Prp-TDP43 A315T mice essential to establish the colony and to guarantee its maintenance, given the effects of the highly aggressive phenotype of this Tg line. As such, in this study we describe our findings in the characterization of the reproductive parameters of Prp-TDP43 A315T mice during four generations. Our results described for the first time, a greater heritage of genetic strengths of TDP-43 A315T transgene by Tg Prp-TDP43 A315T male mice compared to females, even when hemizygous male mice for this GEMs strain have a significantly shorter breeding window than females. Our results may help to redefine strategies aimed at reducing the number of animals required on research.

Animals
Transgenic (Tg) Prp-TDP43 A315T mice [9] and non-Tg C57Bl6/J control mice were used in this study. Animals were group-housed under standard housing conditions with a 12 h light-dark cycle, and food and water ad libitum. Tg Prp-TDP43 A315T mice were fed jellified food in pellet form (D5K52 food. Rettenmaier Ibérica, Spain) to mitigate the intestinal dysmotility phenotype and sudden death [16]. Their general heath was regularly checked as well. All experimental procedures were approved by the Animal Ethics Committee of the National Hospital for Paraplegics (HNP) (Approval No 26/OH 2018) in accordance with the Spanish Guidelines for the Care and Use of Animals for Scientific Purposes.

Colony Amplification for Experimental Animal Generation
Mice hemizygous for TDP-43 A315T transgene are viable and express a mutant human TAR DNA binding protein TDP-43) cDNA harboring an N-terminal Flag tag and an A315T amino acid substitution associated with ALS [9]. This genetic modification is not expected to alter the breeding performance. Tg Prp-TDP43 A315T mice breeding pair were obtained from the Jackson Laboratory (Strain No. 010700, Bar Harbor, ME, USA) to establish the colony. For colony amplification, breeding was arranged between two Tg Prp-TDP43 A315T male mice and non-Tg females on a C57Bl6/J pure background. Then, as hemizygous female carriers live significantly longer than hemizygous males, both female and male Tg Prp-TDP43 A315T mice were backcrossed to C57Bl6/J for four generations for colony maintenance. All animals expressing TDP-43 A315T transgene were confirmed via PCR according to the distributor's protocol.

Statistical Analysis
All values are expressed as the mean ± mean standard deviation (SEM). All statistical analysis was performed using GraphPad Prism (version 8.4.2; San Diego, CA, USA). The parametric two tailed student t test was used for the comparisons between mean groups followed by F test in order to test the homogeneity of variances.

Colony Amplification for Experimental Animal Generation of Tg Prp-TDP43 A315T Mice
Breeder pairs were ordered from Jackson Laboratory (Bar Harbor, ME, USA) to establish a colony at the animal facilities of the HNP (Spain). For the initial colony amplification, mating was arranged between two breeder pair Tg Prp-TDP43 A315T male mice (called F0) delivered by Jackson Laboratory, and two C57Bl6/J females, with the goal of maintaining the same background. Subsequently, both F1 offspring (n = 9 mice in total) were subjected to genotype analysis 21 days after birth. Followed by DNA isolation and PCR analysis (data not showed), our results confirmed only four mice expressed TDP-43 A315T transgene (2 males and 2 females), from a total of 4 females and 5 males pups tested ( Figure 1A). Therefore, only 44% of the F1 progeny were hemizygous for Prp-TDP43 A315T transgene. Values (mean ± SEM) are shown in Table S1.
Then, two Tg Prp-TDP43 A315T mice (2 male and 2 female), from F1 respectively, were backcrossed to 8 C57Bl6/J mice. From this second generation (F2) we obtained 8 litters, one per each Tg Prp-TDP43 A315T female mice, and three per each Tg Prp-TDP43 A315T male mice. Interestingly, following PCR assays, our analysis indicated that 80% of the progeny (8 positive mice/10 mice tested) were positive for the transgene when Tg Prp-TDP43 A315T female mice were backcrossed to C57Bl6/J male mice ( Figure 1B(B1)). This is compared to the 52% of mice expressing TDP-43 A315T transgene when hemizygous Prp-TDP43 A315T male mice were used as breeders (23 positive mice/ 44 mice tested) ( Figure 1B(B2)). Values (mean ± SEM) are shown in Table S2. Therefore, with these results, and considering that it is considerably more difficult to maintain the colony using Tg Prp-TDP43 A315T male mice, the third generation (F3) breeding was arranged between 13 Tg Prp-TDP43 A315T female mice and 1 C57Bl6/J male mice. Surprisingly, although we obtained 15 litters from Tg Prp-TDP43 A315T female mice with 100 mouse pups, following PCR assays (data not showed), our results confirmed 82 non-Tg mice and only 18 mice (13 females and 5 males) were positive for TDP-43 A315T transgene (Figure 2A(A1)). Additionally, one Tg Prp-TDP43 A315T male mice were backcrossed to one C57Bl6/J female mice. Following PCR assays of the offspring (n = 9 mice in total), our analysis indicated 4 positive mice for the transgene, 3 males and 1 female (Figure 2A(A2)). Notably, this result indicates that heritability of the TDP-43 A315T transgene were decreased from the 80% to 17% from F2 to F3 when Tg Prp-TDP43 A315T female mice were backcrossed with C57Bl6/J male mice, while the heritability of the TDP-43 A315T transgene was 44% when hemizygous males were used as breeders (Figure 2A(A2)). Values (mean ± SEM) are shown in Table S3.  To obtain the F4, 7 females and 2 males Tg Prp-TDP43 A315T mice, respectively, were backcrossed to 28 C57Bl6/J mice. In this F4, we obtained 28 litters with a total 189 pups. After genotype procedures, 100 mice were non-Tg mice, and only 89 mice (47 females and 42 males) were positive for TDP-43 A315T transgene by PCR assays. Our data indicated that the 41% of the progeny (21/ 51 pups tested) were positive for TDP-43 A315T transgene when Tg Prp-TDP43 A315T female mice were backcrossed to C57Bl6/J male mice ( Figure 2B(B1)), compared to the 49% (68/ 138 pups tested) of mice expressing TDP-43 A315T transgene when hemizygous Prp-TDP43 A315T male mice were used as breeders ( Figure 2B(B2)). Values (mean ± SEM) are shown in Table S4. Therefore, in total, we obtained 24 litters when Tg Prp-TDP43 A315T female mice were backcrossed to C57Bl6/J male mice, with a total number of 161 offspring, and 47 Tg Prp-TDP43 A315T mice (30% of the progeny) y 30 litters when Tg Prp-TDP43 A315T male mice were backcrossed to C57Bl6/J female, with a total number of 200 offspring, and 99 Tg Prp-TDP43 A315T mice (50% of the progeny) ( Table 1). The mean number pups per litter was similarly independent of the sex of the Tg Prp-TDP43 A315T mice used. However, our data indicated a greater heritage genetic strengths of TDP-43 A315T transgene by Tg Prp-TDP43 A315T male mice compared to female.

Discussion
In recent years, there have been great advances in the use of genetically modified mice to study pathophysiological mechanisms involved in ALS [2]. GEMs have been the predominant experimental tools, and have allowed rapid progress in our understanding of the role of several proposed gene mechanisms in ALS pathogenesis [17]. In general, when working GEMs lines that remain in heterozygosity, it is important to know reproductive parameters in order to design reproduction strategies that guarantee the maximum possible number of animals carrying the desired transgene [18]. However, investigators often avoid properly evaluating this aspect in the favor their usefulness for understanding gene function and genetic basis of diseases. Consequently, very few studies analyze the reproductive parameters of GEMs line. To our knowledge, thus far no study has previously evaluated this aspect in Prp-TDP43 A315T murine models of FTLD/ALS TDP-43 proteinopathy. In this context, we analyzed the reproductive parameters of mice hemizygous for TDP-43 A315T transgene, which were viable and fertile. Our results confirmed a greater heritage of genetic strengths of TDP-43 A315T transgene by Tg Prp-TDP43 A315T male mice compared to females, which may help to redefine strategies aimed at reducing the number of animals required on ALS research.
In our study, both female and male Tg Prp-TDP43 A315T mice were backcrossed to C57Bl6/J pure background during four consecutive generations. The Tg offspring genotype were confirmed by PCR assays. In total, we analyzed 54 litters, with a variable number of pups per each birth, 30 of them obtained when 7 Tg Prp-TDP43 A315T male mice were backcrossed to C57Bl6/J female, and 24 when 21 Tg Prp-TDP43 A315T female mice were backcrossed to C57Bl6/J male, respectively. A total of 361 pups were weaned between 21 and 26 days of age. In all cases, we collected the samples for genotyping at the time of weaning. Following PCR assays, our results confirmed important differences in the number of positive mice for TDP-43 A315T transgene, depending on the sex of the progenitor used to backcrossing with non-Tg C57Bl6/J controls. Indeed, the 49.50% of mice expressing TDP-43 A315T transgene were positive when Tg Prp-TDP43 A315T male mice were backcrossed to C57Bl6/J female, compared to the 19% when Tg Prp-TDP43 A315T female mice were used. Therefore, our study suggests a greater heritage genetic strengths of TDP-43 A315T transgene by Tg Prp-TDP43 A315T male mice compared to females, even when hemizygous male mice for this GEMs strain have a significantly shorter breeding window.
In this context, our study may help to redefine strategies for the experimental use of Tg Prp-TDP43 A315T mice with the aim to reduce the number of animals required on research. This is important as in basic research with animal models the focus has been on males and excluded females [19]. In the field of our study, and in general in neuroscience, when researchers use GEMs mice, males are preferred in order to avoid the effects of the estrous cycle on the experimental results [20][21][22][23]. Indeed, the sex of animal subjects is omitted in 22-42% of articles in neuroscience, and results of studies in males are often generalized to females without justification. This presents a problem as, in many cases, including our study, sex has proven an important variable to take in consideration.
In summary, our study is the first to describe importance of reproductive parameters of Tg Prp-TDP43 A315T mice. Our findings indicate that investigators need to redefine strategies for the experimental use of Tg Prp-TDP43 A315T mice as gender differences is an important aspect of breeding to take into consideration to ensure the principle of reduction in animal experimentation. In addition, our data will also significantly aid researchers to ensure and reduce the general maintenance cost of the colony, including animal facility laboring costs, less sampling for genotyping and, subsequently, fewer negative animals will be slaughtered. Nonetheless, future studies will be necessary to determine not only the genetic mechanism involved in the heritability for TDP-43 A315T transgene, but also to further study the contribution of the nutrition as a key factor for the genetic inheritance on this murine model of FTLD/ALS TDP-43 proteinopathy.  Funding: This work was supported by the funding from the Consejería de Educación, Cultura y Deportes, Fondo Europeo de Desarrollo Regional (FEDER), Junta de Comunidades de Castilla-La Mancha (SBPLY/17/180501/000303).