Abstract
ABSTRACTA gene of interest can be efficiently modified using transcription activator-like effector nucleases (TALENs) (Christian et al., 2010;Li et al., 2011). However, if a target gene is essential for development, growth and fertility, use of TALENs with high mutagenic activity in F0 frogs could result in developmental disorders or sterility, which would reduce the number of F1 progeny and make F1 phenotypical analysis difficult. We used the 3′ untranslated region of DEADSouth gene (DS-3′) of Xenopus tropicalis to solve this problem, because the addition of the DS-3′ to mRNA is known to induce primordial germ cell (PGC)-specific expression and reduce the stability in somatic cells of mRNA in Xenopus laevis. At first, we inserted the X. tropicalis DS-3′ downstream of the EGFP termination codon and confirmed that the EGFP expression was specifically detected in PGCs for three weeks. Therefore, we inserted the DS-3′ downstream of the termination codon of the TALEN coding sequence. The tyrosinase gene was selected as the target gene for TALEN because the bi-allelic mutation of this gene is easily discernible by the albino phenotype. When fertilized eggs were microinjected with TALEN mRNAs fused to the DS-3′, their sperm and oocytes had a high rate (84–100%) of target-gene modification in contrast to the lower rate (0–45%) of nucleotide alteration observed in somatic cells.
Highlights
Targeted gene disruption is becoming a common, facile and essential method for demonstrating the function of a specific gene and is currently performed using transcription activator-like effector nucleases (TALENs) (Cermak et al, 2011; Christian et al, 2010; Li et al, 2011) or the CRISPR/Cas system (Jinek et al, 2012)
We hypothesized that injecting TALEN mRNAs fused to the DS39 would result in primordial germ cell (PGC)-specific TALEN expression
EGFP fluorescence was frequently observed in the PGCs in the mesentery of eight-day-old tadpoles (19 EGFPpositive tadpoles on the eighth day out of 39 tadpoles injected with EGFP-DS mRNA) and in the genital ridges of 21-day-old tadpoles (13 EGFP-positive tadpoles on the 21st day out of 13 EGFP-positive tadpoles on the eighth day)
Summary
Targeted gene disruption is becoming a common, facile and essential method for demonstrating the function of a specific gene and is currently performed using transcription activator-like effector nucleases (TALENs) (Cermak et al, 2011; Christian et al, 2010; Li et al, 2011) or the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPRassociated proteins) system (Jinek et al, 2012). TALENs are fusion proteins, consisting of a nuclear localization signal, a target DNA-binding domain and the nuclease domain of FokI, that enter the nucleus and recognize 15–24 nucleotides of both the left and right halves of the target sequence to form a dimer in the Division of Embryology and Genetics, Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashihiroshima 739-8526, Japan. We succeeded in preferentially editing the genome of the germ cells by injecting TALEN mRNAs fused to the DS-39 into Xenopus embryos
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