RESUMO
Parental behavioural traits can be transmitted by non-genetic mechanisms to the offspring. Although trait transmission via sperm has been extensively researched, epidemiological studies indicate the exclusive/prominent maternal transmission of many non-genetic traits. Since maternal conditions impact the offspring during gametogenesis and through fetal/early-postnatal life, the resultant phenotype is likely the aggregate of consecutive germline and somatic effects; a concept that has not been previously studied. Here, we dissected a complex maternally transmitted phenotype, reminiscent of comorbid generalized anxiety/depression, to elementary behaviours/domains and their transmission mechanisms in mice. We show that four anxiety/stress-reactive traits are transmitted via independent iterative-somatic and gametic epigenetic mechanisms across multiple generations. Somatic/gametic transmission alters DNA methylation at enhancers within synaptic genes whose functions can be linked to the behavioural traits. Traits have generation-dependent penetrance and sex specificity resulting in pleiotropy. A transmission-pathway-based concept can refine current inheritance models of psychiatric diseases and facilitate the development of better animal models and new therapeutic approaches.
Assuntos
Comportamento Animal/fisiologia , Epigênese Genética , Células Germinativas/fisiologia , Herança Materna/fisiologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Ansiedade/genética , Ansiedade/psicologia , Metilação de DNA/genética , Modelos Animais de Doenças , Feminino , Gametogênese/fisiologia , Impressão Genômica/fisiologia , Hipotermia/induzido quimicamente , Hipotermia/genética , Hipotermia/psicologia , Masculino , Metabolômica/métodos , Camundongos , Camundongos Knockout , Modelos Animais , Penetrância , Fenótipo , Receptor 5-HT1A de Serotonina/genética , Receptor 5-HT1A de Serotonina/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Estresse Psicológico/genética , Estresse Psicológico/psicologiaRESUMO
The genomes of more than 50 organisms have now been manipulated due to rapid advancement of gene editing technology. One way to perform gene editing in the mouse using the CRISPR/CAS system, guide RNA (gRNA) and CAS9 mRNA transcribed in vitro are microinjected into fertilized eggs that are then allowed to develop to term. As a rule, gRNAs are tested first in tissue culture cells and the one with the highest locus-specific cleavage activity is chosen for microinjection. For cell transfections, gRNAs are typically expressed using the human U6 promoter (hU6). However, gRNAs for microinjection into zygotes are obtained by in vitro transcription from a T7 bacteriophage promoter in a separate plasmid vector. Here, we describe the design and construction of a combined U6T7 hybrid promoter from which the same gRNA sequence can be expressed. An expression vector containing such a hybrid promoter can now be used to generate gRNA for testing in mammalian cells as well as for microinjection purposes. The gRNAs expressed and transcribed from this vector are found to be functional in cells as well as in mice.
