Simple knockout by electroporation of engineered endonucleases into intact rat embryos.

Takehito Kaneko,Takashi Yamamoto,Tetsushi Sakuma,Tomoji Mashimo

doi:10.1038/srep06382

Takehito Kaneko, Takashi Yamamoto + Show 2 more

Open Access

https://doi.org/10.1038/srep06382

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Journal: Scientific Reports	Publication Date: Oct 1, 2014
Citations: 185	License type: CC BY 4.0

Affiliation: Kyoto University, Hiroshima University

Abstract

Engineered endonucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, provide a powerful approach for genome editing in animals. However, the microinjection of endonucleases into embryos requires a high skill level, is time consuming, and may cause damage to embryos. Here, we demonstrate that the electroporation of endonuclease mRNAs into intact embryos can induce editing at targeted loci and efficiently produce knockout rats. It is noteworthy that the electroporation of ZFNs resulted in an embryonic survival rate (91%) and a genome-editing rate (73%) that were more than 2-fold higher than the corresponding rates from conventional microinjection. Electroporation technology provides a simple and effective method to produce knockout animals.

Highlights

Introduction of the zinc-finger nucleases (ZFNs) ortranscription activator-like effector nucleases (TALENs) plasmid into fibroblast-like cells
ZFN, TALEN, or clustered regularly interspaced short palindromic repeat (CRISPR) mRNA was electroporated into embryos (Fig. 1a)
Pronuclear-stage embryos were placed in a line on the glass chamber between metal plates that were filled with phosphate-buffered saline (PBS) containing mRNA (Fig. 1a). mRNAs were efficiently introduced into intact embryos with a 3-step pulse system (Fig. 1b)

Summary

Results

ZFN, TALEN, or CRISPR mRNA was electroporated into embryos (Fig. 1a). Pronuclear-stage embryos were placed in a line on the glass chamber between metal plates that were filled with phosphate-buffered saline (PBS) containing mRNA (Fig. 1a). MRNAs were efficiently introduced into intact embryos with a 3-step pulse system (Fig. 1b). Several of the first transfer pulses transfer mRNA into the cytoplasm. The polarity-changed second transfer pulse increases the chance of transferring mRNA into embryos (Fig. 1c). Introduction of tetramethylrhodamine-labelled dextran into intact pronuclear-stage embryos. To test the electroporation conditions to introduce materials into embryos, tetramethylrhodamine-labelled dextran (3 kDa molecular weight), which is visualised and nontoxic to living cells, was used. Pronuclear-stage embryos were collected from superovulated female rats the day after mating.

CRISPR Microinjection

Discussion

Additional information