Abstract
An insulin-like growth factor-1 receptor (IGF1R) variant in exon 6 (Arg-407-His) in Ashkenazi Jewish centenarians was previously found to be associated with reduced IGF1R activity. To further study this longevity associated IGF1R variant, we generated a novel mouse line carrying the R407H variant in exon 6 of the Igf1r gene by employing CRISPR/Cas9 genome editing technology. Here, we show that the Igf1r gene can be edited in mouse embryos by zygotic electroporation of Cas9 protein and a single-guide RNAs together with a single stranded oligonucleotide donor containing the desired key nucleotide changes at the Igf1r locus. Sequence analysis of F0 and F1 mice following targeted editing demonstrated the robustness of this approach in mice using CRISPR/Cas9 directed homologous recombination (HDR). Western blot analysis indicates that mice heterozygous for the variant have a significant decrease in IGF1R phosphorylation in various tissues, including skeletal muscle, compared to wildtype. In addition, depletion of IGF1R signaling specifically in skeletal muscle of progeroid Ercc1 -/Δ mice resulted in extended health span and median lifespan providing the rationale for long term lifespan studies in Igf1r hR407H variant mice. This mouse line will be a valuable genetic tool to help determine the impact of IGF1R signaling on aging and longevity. The CRISPR editing approach represents a prototype for generating additional longevity associated gene variant mouse lines to study relevance to human exceptional longevity.
Highlights
Loss of function in genes encoding components of insulin/insulinlike growth factor-1 (IGF1) signaling have been shown to extend lifespan in yeast, worms, flies, and mice [1]
We generated a mouse line that replicates the human insulin-like growth factor-1 receptor (IGF1R) R407H longevity-related variant observed in Ashkenazi Jewish centenarians using CRIPSR/Cas9 technology to generate an R408H substitution in the mouse Igf1r gene
The first step was electroporation of the RNP consisting of the sgRNAIgf1r and Streptococcus pyogenes Cas9 (SpCas9) mRNA with a 123-bp single stranded oligonucleotide donor template into one-cell mouse zygotes
Summary
Loss of function in genes encoding components of insulin/insulinlike growth factor-1 (IGF1) signaling have been shown to extend lifespan in yeast, worms, flies, and mice [1]. While insulin mainly provides short-term effects that regulate metabolic pathways, IGF1 promotes chronic effects that determine cell fates, such as growth or differentiation of target cells, cell survival and maintenance of cell function [2]. Studies have reported on levels of IGF1 and related molecules in centenarians, but these lacked adequate controls This shortcoming was overcome by establishing a cohort of Ashkenazi Jewish decent with extended longevity and their offspring, and age- and sex-matched controls without a family history of unusual longevity [3]. Using this group of individuals, variants in the IGF1R were found to be enriched in centenarians compared to controls. In a follow up study, engineered to express the two variants in Igf1r-/- mouse embryonic fibroblasts (MEFs) showed reduced phosphorylation of IGF1R [4]
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