RESUMO
The laboratory mouse is the most used animal model in biomedical research. Several artificial reproductive techniques, such as revitalization of cryopreserved strains, rederivation after hygienic contaminations and the production of transgenic mouse models, require the transfer of preimplantation embryos to surrogate mothers. Pseudopregnancy is essential in recipient females and is induced by mating with sterile males. Commonly, surgically vasectomized males are used for this purpose. As an alternative, genetically modified mouse strains have been identified, in which homozygous infertile males are sexually active. Here, we investigated the suitability of genetically infertile Gapdhstm1Dao males under routine laboratory conditions with respect to plug rates, pregnancy rates and frequency of born offspring after embryo transfer. Our results showed no significant differences for these aspects between Gapdhstm1Dao and vasectomized CD2F1 males. In addition, we evaluated the efforts to obtain a defined number of sterile males either by breeding of sterile mutants or surgical vasectomy, and addressed the impact of both options on animal welfare. In conclusion, infertile males of the Gapdhstm1Dao line are a reliable alternative to vasectomized males for the induction of pseudopregnancy, and can contribute to the refinement of the procedure by avoiding surgical interventions.
Assuntos
Transferência Embrionária/veterinária , Infertilidade Masculina/veterinária , Camundongos Knockout , Modelos Animais , Pseudogravidez/veterinária , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Gravidez , Taxa de Gravidez , VasectomiaRESUMO
Chemical random mutagenesis techniques with the germ line supermutagen N-ethyl-N-nitrosourea (ENU) have been established to provide comprehensive collections of mouse models, which were then mined and analyzed in phenotype-driven studies. Here, we applied ENU mutagenesis in a high-throughput fashion for a gene-driven identification of new mutations. Selected members of the large superfamily of G protein-coupled receptors (GPCR), melanocortin type 3 (Mc3r) and type 4 (Mc4r) receptors, and the orphan chemoattractant receptor GPR33, were used as model targets to prove the feasibility of this approach. Parallel archives of DNA and sperm from mice mutagenized with ENU were screened for mutations in these GPCR, and in vitro assays served as a preselection step before in vitro fertilization was performed to generate the appropriate mouse model. For example, mouse models for inherited obesity were established by selecting fully or partially inactivating mutations in Mc4r. Our technology described herein has the potential to provide mouse models for a GPCR dysfunction of choice within <4 mo and can be extended to other gene classes of interest.
Assuntos
Modelos Animais de Doenças , Etilnitrosoureia/toxicidade , Mutação/genética , Receptores Acoplados a Proteínas G/genética , Alquilantes/toxicidade , Animais , Células COS , Chlorocebus aethiops , Análise Mutacional de DNA/métodos , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutagênese/efeitos dos fármacos , Filogenia , Receptor Tipo 3 de Melanocortina/genética , Receptor Tipo 3 de Melanocortina/fisiologia , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
The SMA1-mouse is a novel ethyl-nitroso-urea (ENU)-induced mouse mutant that carries an a-->g missense mutation in exon 5 of the GH gene, which translates to a D167G amino acid exchange in the mature protein. Mice carrying the mutation are characterized by dwarfism, predominantly due to the reduction (sma1/+) or absence (sma1/sma1) of the GH-mediated peripubertal growth spurt, with sma1/+ mice displaying a less pronounced phenotype. All genotypes are viable and fertile, and the mode of inheritance is in accordance with a semidominant Mendelian trait. Adult SMA1 mice accumulate excessive amounts of sc and visceral fat in the presence of elevated plasma ghrelin levels, possibly reflecting altered energy partitioning. Our results suggest impaired storage and/or secretion of pituitary GH in mutants, resulting in reduced pituitary GH and reduced GH-stimulated IGF-1 expression. Generation and identification of the SMA1 mouse exemplifies the power of the combination of random mouse mutagenesis with a highly detailed phenotype-analysis as a successful strategy for the detection and analysis of novel gene-function relationships.
Assuntos
Nanismo/genética , Hormônio do Crescimento/genética , Mutação de Sentido Incorreto , Obesidade/genética , Hormônios Peptídicos/sangue , Tecido Adiposo , Sequência de Aminoácidos , Animais , Composição Corporal/genética , Peso Corporal/genética , Éxons , Feminino , Fertilidade , Ligação Genética , Genótipo , Grelina , Hormônio do Crescimento/análise , Hormônio do Crescimento/química , Humanos , Fator de Crescimento Insulin-Like I/análise , Fator de Crescimento Insulin-Like I/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Fenótipo , Hipófise/química , Alinhamento de SequênciaRESUMO
Degenerative disorders of motor neurons include a range of progressive fatal diseases such as amyotrophic lateral sclerosis (ALS), spinal-bulbar muscular atrophy (SBMA), and spinal muscular atrophy (SMA). Although the causative genetic alterations are known for some cases, the molecular basis of many SMA and SBMA-like syndromes and most ALS cases is unknown. Here we show that missense point mutations in the cytoplasmic dynein heavy chain result in progressive motor neuron degeneration in heterozygous mice, and in homozygotes this is accompanied by the formation of Lewy-like inclusion bodies, thus resembling key features of human pathology. These mutations exclusively perturb neuron-specific functions of dynein.