Abstract
Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.
Highlights
Programmable nucleases such as transcription activator-like effector nuclease (TALEN) and CRISPR/Cas9 have been used to efficiently produce gene knock-out animals in various species including those previously regarded as non-permissive by conventional gene disruption methods [1,2,3,4,5,6,7,8,9]
These results suggest that the injection of tyr TALEN mRNAs into germinal vesicle (GV) oocytes enables the full albino phenotype in the F0 generation
The injection of TALEN mRNAs from the GV oocyte stage reduced unedited cells in F0 embryos as judged by tyr knock-out, and the embryonic lethal phenotype of pax6 knock-out was recapitulated in most of F0 embryos. These strong knock-out phenotypes seem to be caused by the presence of TALEN proteins by the time of fertilization, which allows instant access to zygotic chromatin and to chromatin of embryos at early cleavage stages
Summary
Programmable nucleases such as TALENs and CRISPR/Cas have been used to efficiently produce gene knock-out animals in various species including those previously regarded as non-permissive by conventional gene disruption methods [1,2,3,4,5,6,7,8,9]. A pair of customized TALENs (right and left side TALENs) binds to their target sites and Fok I is dimerized to introduce DNA double strand breaks. This is followed by DNA repair mainly mediated by non-homologus end joining, resulting in deletion and/or insertion mutations in targeted genes. Such TALEN activity has recently been enhanced by modifying repeat sequences in the TALE DNA-binding modules, referred to as Platinum TALEN [15]. It has recently been shown that Platinum TALEN fits well with the efficient gene knock-in system based on microhomology-mediated end-joining at the TALEN-targeted site [19]
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