RESUMEN
Autism spectrum disorder (ASD) is a neurodevelopmental disorder with core symptoms that include poor social communication, restricted interests, and repetitive behaviors. Several ASD mouse models exhibit impaired social interaction, anxiety-like behavior, and elevated perseveration. Large-scale whole exome sequencing studies identified many genes putatively associated with ASD. Like chromodomain helicase DNA binding protein 8 (CHD8), the most frequently mutated gene in individuals with ASD, the candidate gene AT-rich interaction domain 1B (ARID1B) encodes a chromatin remodeling factor. Arid1b heterozygous knockout (hKO) mice exhibited ASD-like traits related to social behavior, anxiety, and perseveration, in addition to associated features reported in some cases of ASD, such as reduced weight, impaired motor coordination, and hydrocephalus. Hydrocephalus was present in 5 of 91 hKO mice, while it was not observed in wild-type littermates (0 of 188). Genome-wide gene expression patterns in Arid1b hKO mice were similar to those in ASD patients and Chd8-haploinsufficient mice, an ASD model, and to developmental changes in gene expression in fast-spiking cells in the mouse brain. Our results suggest that Arid1b haploinsufficiency causes ASD-like phenotypes in mice.
Asunto(s)
Trastorno del Espectro Autista/genética , Proteínas de Unión al ADN/genética , Haploinsuficiencia/genética , Factores de Transcripción/genética , Animales , Trastorno del Espectro Autista/fisiopatología , Conducta Animal , Ensamble y Desensamble de Cromatina/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Humanos , Hidrocefalia/genética , Hidrocefalia/fisiopatología , Ratones , Ratones NoqueadosRESUMEN
Conditional knockout using Cre/lox is essential for functional analysis of genes. CRISPR/Cas in combination with two sets of guide RNAs and a single-stranded oligonucleotide enables simultaneous insertion of two lox sequences. However, this method induces double-strand breaks at two sites on the same chromosome, which causes an undesirable chromosomal deletion and reduces the flanked lox (flox) rate. To solve this problem, we investigated a method that sequentially introduces each lox sequence at the 1-cell and 2-cell embryonic stages, respectively. The sequential method was applied to both microinjection and electroporation systems. Sequential electroporation improved the flox efficiency compared with ordinary simultaneous microinjection, leading to a high yield of offspring with floxed alleles. Finally, we directly produced Cre/lox mice containing both the Cre transgene and floxed allele via sequential electroporation using Cre zygotes, which accelerated the generation of conditional knockout mice compared with the ordinary method.