Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host
transfer in Xenopus laevis
#MMPMID25596277
Nakajima K
; Yaoita Y
Biol Open
2015[Jan]; 4
(2
): 180-5
PMID25596277
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Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs)
and the CRISPR/Cas (clustered regularly interspaced short palindromic
repeats/CRISPR-associated proteins) system are potentially powerful tools for
producing tailor-made knockout animals. However, their mutagenic activity is not
high enough to induce mutations at all loci of a target gene throughout an entire
tadpole. In this study, we present a highly efficient method for introducing gene
modifications at almost all target sequences in randomly selected embryos. The
gene modification activity of TALEN is enhanced by adopting the host-transfer
technique. In our method, the efficiency is further improved by injecting TALEN
mRNAs fused to the 3'UTR of the Xenopus DEADSouth gene into oocytes, which are
then transferred into a host female frog, where they are ovulated and fertilized.
The addition of the 3'UTR of the DEADSouth gene promotes mRNA translation in the
oocytes and increases the expression of TALEN proteins to near-maximal levels
three hours post fertilization (hpf). In contrast, TALEN mRNAs without this 3'UTR
are translated infrequently in oocytes. Our data suggest that genomic DNA is more
sensitive to TALEN proteins from fertilization to the midblastula (MBT) stage.
Our method works by increasing the levels of TALEN proteins during the pre-MBT
stages.
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