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Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis.

ABSTRACT: 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.

SUBMITTER: Nakajima K

PROVIDER: S-EPMC4365486 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis.

Nakajima Keisuke K Yaoita Yoshio Y

Biology open 20150116 2

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 ...[more]

PMID: 25596277

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Project description:BACKGROUND:A new type of influenza virus, known as type D, has recently been identified in cattle and pigs. Influenza D virus infection in cattle is typically asymptomatic; however, its infection in swine can result in clinical disease. Swine can also be infected with all other types of influenza viruses, namely A, B, and C. Consequently, swine can serve as a "mixing vessel" for highly pathogenic influenza viruses, including those with zoonotic potential. Currently, the only antiviral drug available targets influenza M2 protein ion channel is not completely effective. Thus, it is necessary to develop an M2 ion channel blocker capable of suppressing the induction of resistance to the genetic shift. To provide a basis for developing novel ion channel-blocking compounds, we investigated the properties of influenza D virus M2 protein (DM2) as a drug target. RESULTS:To test the ion channel activity of DM2, the DNA corresponding to DM2 with cMyc-tag conjugated to its carboxyl end was cloned into the shuttle vector pNCB1. The mRNA of the DM2-cMyc gene was synthesized and injected into Xenopus oocytes. The translation products of DM2-cMyc mRNA were confirmed by immunofluorescence and mass spectrometry analyses. The DM2-cMyc mRNA-injected oocytes were subjected to the two-electrode voltage-clamp (TEVC) method, and the induced inward current was observed. The midpoint (Vmid) values in Boltzmann modeling for oocytes injected with DM2-cMyc RNA or a buffer were -152 and -200 mV, respectively. Assuming the same expression level in the Xenopus oocytes, DM2 without tag and influenza C virus M2 protein (CM2) were subjected to the TEVC method. DM2 exhibited ion channel activity under the condition that CM2 ion channel activity was reproduced. The gating voltages represented by Vmid for CM2 and DM2 were -141 and -146 mV, respectively. The reversal potentials observed in ND96 for CM2 and DM2 were -21 and -22 mV, respectively. Compared with intact DM2, DM2 variants with mutation in the YxxxK motif, namely Y72A and K76A DM2, showed lower Vmid values while showing no change in reversal potential. CONCLUSION:The M2 protein from newly isolated influenza D virus showed ion channel activity similar to that of CM2. The gating voltage was shown to be affected by the YxxxK motif and by the hydrophobicity and bulkiness of the carboxyl end of the molecule.

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Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis.

Publications

Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis.

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