RESUMO
PURPOSE: The purpose of this study was to establish a GAL4/VP16-based binary transactivation system that was active in the lens and corneal epithelium of transgenic mice. METHODS: We generated transgenic mice with the transcriptional transactivator GAL4/VP16 driven by a modified Pax6 promoter that is active in lens and corneal epithelial cells. We also generated and tested UAS-lacZ reporter mice. Wild type and transgenic mice were analyzed by histological, in situ, and Southern hybridization techniques. RESULTS: Five families (OVE1931, OVE1934, OVE1935, OVE1936, and OVE1937) that carry the Pax6-GAL4/VP16 transgene were generated. Unexpectedly, mice from three of the transgenic lines showed ocular abnormalities. In the family OVE1936, cataracts were seen in the heterozygous mice at the time of eyelid opening and homozygotes showed microphthalmia. Transgenic mice in families OVE1931 and OVE1937 appeared normal. Histological analysis of ocular sections of OVE1934, OVE1935, and OVE1936 homozygous transgenic mice showed intracorneal positioning of the lens. The corneal stromal cells were disorganized and there was no distinctive corneal endothelial layer. In situ hybridizations showed robust expression of the GALVP16 transgene in the lens and corneal epithelial cells of the OVE1934, OVE1935, and OVE1936, but not in OVE1931 or OVE1937 families. Bigenic embryos generated by mating the Pax6-GAL4/VP16 mice to the UAS-lacZ mice showed that the GAL4/VP16 transgenic protein is functional and can induce eye-specific expression of a UAS-lacZ reporter gene. CONCLUSIONS: Our data suggest that (1) expression the GAL4/VP16 transgene induces changes in gene expression in lens cells, (2) that developmentally important genes are affected, and (3) that bigenic phenotypes will need to be interpreted with caution.
Assuntos
Córnea/anormalidades , Anormalidades do Olho/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Cristalino/anormalidades , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Transativadores/genética , Ativação Transcricional/fisiologia , Animais , Southern Blotting , Catarata/genética , Catarata/metabolismo , Catarata/patologia , Córnea/metabolismo , Córnea/patologia , Doenças da Córnea/genética , Doenças da Córnea/metabolismo , Doenças da Córnea/patologia , Anormalidades do Olho/metabolismo , Anormalidades do Olho/patologia , Proteínas do Olho/metabolismo , Feminino , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Cristalino/metabolismo , Cristalino/patologia , Masculino , Camundongos , Camundongos Transgênicos , Microftalmia/genética , Microftalmia/patologia , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Transgenes , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Autism spectrum disorder (ASD) diagnoses are behaviorally based with no defined universal biomarkers, occur at a 1:110 ratio in the population, and predominantly affect males compared to females at approximately a 4:1 ratio. One approach to investigate and identify causes of ASD is to use organisms that display abnormal behavioral responses that model ASD-related impairments. This study describes a novel transgenic mouse, MALTT, which was generated using a forward genetics approach. It was determined that the transgene integrated within a non-coding region on the X chromosome. The MALTT line exhibited a complete repertoire of ASD-like behavioral deficits in all three domains required for an ASD diagnosis: reciprocal social interaction, communication, and repetitive or inflexible behaviors. Specifically, MALTT male mice showed deficits in social interaction and interest, abnormalities in pup and juvenile ultrasonic vocalization communications, and exhibited a repetitive stereotypy. Abnormalities were also observed in the domain of sensory function, a secondary phenotype prevalently associated with ASD. Mapping and expression studies suggested that the Fam46 gene family may be linked to the observed ASD-related behaviors. The MALTT line provides a unique genetic model for examining the underlying biological mechanisms involved in ASD-related behaviors.
Assuntos
Agressão/psicologia , Transtorno Autístico/psicologia , Modelos Animais de Doenças , Comportamento Social , Análise de Variância , Animais , Transtorno Autístico/genética , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Filtro Sensorial , Comportamento Estereotipado , Vocalização AnimalRESUMO
The transgenic mouse line OVE459 carries a transgene-induced insertional mutation resulting in autosomal recessive congenital hydrocephalus. Homozygous transgenic animals experience ventricular dilation with perinatal onset and are noticeably smaller than hemizygous or non-transgenic littermates within a few days after birth. Fluorescence in situ hybridization (FISH) revealed that the transgene inserted in a single locus on mouse Chromosome (chr) 8, region D2-E1. Genetic crosses between hemizygous OVE459 mice and mice heterozygous for the spontaneous mutation hydrocephalus-3 (hy3) produced hydrocephalic offspring with a frequency of 22%, demonstrating that these two mutations are allelic. A genomic library was made by using DNA from homozygous OVE459 mice, and genomic DNA flanking the transgene insertion site was isolated and sequenced. A PCR polymorphism between C57BL/6 DNA and Mus spretus was used to map the location of the transgene insert to 1.06 cM +/- 0.75 proximal to D8Mit152 by using the Jackson Laboratory Backcross DNA Panel Mapping Resource. Furthermore, sequence analysis from a mouse bacterial artificial chromosome (BAC) clone, positive for unique markers on both sides of the transgene insertion site, demonstrated that the genomic DNAs flanking each side of the transgene insertion are physically separated by approximately 51 kb on the wild-type mouse chromosome.