P22 Age and sex related differences in satellite cell number, proliferation and self renewal

Abstract

knockdown of FKRP expression by anti-sense morpholino (MO) injection into zebrafish embryos. This system gives a range of phenotypes but the knockdown has limited duration. A stable mutant line would negate the problems of a transient knockdown system which include decrease of knockdown efficiency due to cell division and consequent dilution of MO with time. Zinc Finger Nucleases (ZFNs) which are artificial nucleases specific to 9 base pairs (bp) target regions either side of a 5–7bp spacer. ZFNs are capable of causing a range of genomic mutations at the target site by inducing double-strand break repair through non-homologous end joining. There are two strategies being adopted, these are Oligomerized Pool Engineering (OPEN) and Context Dependant Assembly (CODA). The sequences generated in each approach for the zinc fingers are cloned into a vector containing the Fok-I nuclease domain. RNA from the expression vectors is injected into the embryo at the single cell stage. Mutant embryos will be screened by genotype against database sequences for mutations. Immunostaining will be used to investigate the phenotypes of the mutants focussing on eyes and muscle.