RESUMO
Mutations and single base pair polymorphisms in various genes have been associated with increased susceptibility to inflammatory bowel disease (IBD). We have created a series of rat strains carrying targeted genetic alterations within three IBD susceptibility genes: Nod2, Atg16l1, and Il23r, using CRISPR/Cas9 genome editing technology. Knock-out alleles and alleles with known human susceptibility polymorphisms were generated on three different genetic backgrounds: Fischer, Lewis and Sprague Dawley. The availability of these rat models will contribute to our understanding of the basic biological roles of these three genes as well as provide new potential IBD animal models.
Assuntos
Predisposição Genética para Doença , Doenças Inflamatórias Intestinais/genética , Proteína Adaptadora de Sinalização NOD2/genética , Receptores de Interleucina/genética , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes , Genótipo , Humanos , Doenças Inflamatórias Intestinais/patologia , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Ratos , Proteínas de Transporte Vesicular/genéticaRESUMO
The Cre/loxP recombination system has revolutionized the ability to genetically manipulate animal genomes in order to conditionally control gene expression. With recent advances in genome editing, barriers to manipulating the rat genome have been overcome and it is now possible to generate new rat strains (Cre drivers) in which Cre recombinase expression is carefully controlled temporally and/or spatially. However, the ability to evaluate and characterize these Cre driver strains is limited by the availability of reliable reporter rat strains. Here, we describe the generation and characterization of a new transgenic rat strain in which conditional expression of the ZsGreen fluorescent protein gene requires the presence of exogenous Cre recombinase. Breeding Cre-expressing rat strains to this stable ZsGreen reporter strain provides an ideal method for validating new rat Cre driver lines and will greatly accelerate the characterization pipeline.
Assuntos
Genes Reporter/genética , Engenharia Genética/métodos , Integrases/genética , Proteínas Luminescentes/genética , Animais , Feminino , Expressão Gênica , Regulação da Expressão Gênica/genética , Genoma/genética , Integrases/biossíntese , Masculino , Ratos , Ratos Endogâmicos F344 , Ratos Transgênicos , Recombinação Genética/genéticaRESUMO
Germline competent embryonic stem (ES) cells can serve as a tool to create genetically engineered rat strains used to elucidate gene function or provide disease models. In optimum culture conditions, ES cells are able to retain their pluripotent state. The type of components present and their concentration in ES cell culture media greatly influences characteristics of ES cells including the ability to maintain the cells in a pluripotent state. We routinely use 2i media containing inhibitors CHIR99021 and PD0325901 to culture rat ES cells. CHIR99021 specifically inhibits the Gsk3ß pathway. We have found that the vendor source of CHIR99021 has a measurable influence on the level of aneuploidy seen over time as rat ES cells are passaged. Karyotyping of three different rat ES cell lines passaged multiple times showed increased aneuploidy when CHIR99021 from source B was used. Mass spectrometry analysis of this inhibitor showed the presence of unexpected synthetic small molecules, which might directly or indirectly cause increases in chromosome instability. Identifying these molecules could further understanding of their influence on chromosome stability and indicate how to improve synthesis of this media component to prevent deleterious effects in culture.
