IADR Abstract Archives
Conditional Notch1-knockout Transgenic Mouse Generation by CRISPR/Cas9 Genome Engineering System
Objectives: Notch1 is a transmembrane receptor involved in the differentiation and proliferation of many embryonic and adult cell types. Notch pathway has been implicated as driving factor in some hematologic malignancy but possess tumor suppressing function in solid tumor e.g. oral squamous cell carcinoma. Previous studies have shown total Notch1 knockout mouse suffered embryonic fatality so we require conditional gene ablation to investigate the role of Notch1. Here we show the preliminary result of tissue-specific Notch1 knockout mouse generated by recent genome engineering technique of CRISPR/Cas9 system.
Methods: We designed a transgene that expresses a single guide RNA targeting the second exon of Notch1 driven by an U6 promoter and Cas9 driven by a Keratin 14 (KRT14) promoter, to give rise to Notch1 knockout (KO) specifically in oral epithelium and skin of the transgenic mice. The linearized transgene was injected to mouse zygotes and transplanted to pseudopregnant female mouse. Delivered full-term mice were analyzed for induced phenotype.
Results: By genotyping, we validated 6 mice as KRT14-driven Notch1 KO mouse founders. Two of these founder mice showed phenotype of ipsilateral patches of almost complete hair loss suggesting genetic mosaicism, visually observable by week eight postnatal. This occured phenotype is in accordance to previous study result of KRT14-Notch1 KO by Cre-lox system. Using DNA extracted from the remaining hair in the hair-loss-patches, we performed direct sequencing and found that these mice have nucleotide base pairs deletion in the targeted Notch1 gene, confirming the insertion or deletion mutation induced by CRISPR/Cas9 system. We continue to follow the development of this KO mouse.
Conclusions: Though more analysis should be conducted to thoroughly establish the phenotype, we have successfully showed a promising new knockout mouse model by applying a recent efficient method of genome engineering.
Methods: We designed a transgene that expresses a single guide RNA targeting the second exon of Notch1 driven by an U6 promoter and Cas9 driven by a Keratin 14 (KRT14) promoter, to give rise to Notch1 knockout (KO) specifically in oral epithelium and skin of the transgenic mice. The linearized transgene was injected to mouse zygotes and transplanted to pseudopregnant female mouse. Delivered full-term mice were analyzed for induced phenotype.
Results: By genotyping, we validated 6 mice as KRT14-driven Notch1 KO mouse founders. Two of these founder mice showed phenotype of ipsilateral patches of almost complete hair loss suggesting genetic mosaicism, visually observable by week eight postnatal. This occured phenotype is in accordance to previous study result of KRT14-Notch1 KO by Cre-lox system. Using DNA extracted from the remaining hair in the hair-loss-patches, we performed direct sequencing and found that these mice have nucleotide base pairs deletion in the targeted Notch1 gene, confirming the insertion or deletion mutation induced by CRISPR/Cas9 system. We continue to follow the development of this KO mouse.
Conclusions: Though more analysis should be conducted to thoroughly establish the phenotype, we have successfully showed a promising new knockout mouse model by applying a recent efficient method of genome engineering.