Successful production of genome-edited rats by the rGONAD method
Friday, 2018/08/24 | 07:46:04
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Tomoe Kobayashi, Masumi Namba, Takayuki Koyano, Masaki Fukushima, Masahiro Sato, Masato Ohtsuka and Makoto Matsuyama BMC Biotechnology 2018 18:19 https://doi.org/10.1186/s12896-018-0430-5 BackgroundRecent progress in development of the CRISPR/Cas9 system has been shown to be an efficient gene-editing technology in various organisms. We recently developed a novel method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) in mice; a novel in vivo genome editing system that does not require ex vivo handling of embryos, and this technology is newly developed and renamed as “improved GONAD” (i-GONAD). However, this technology has been limited only to mice. Therefore in this study, we challenge to apply this technology to rats. ResultsHere, we determine the most suitable condition for in vivo gene delivery towards rat preimplantation embryos using tetramethylrhodamine-labelled dextran, termed as Rat improved GONAD (rGONAD). Then, to investigate whether this method is feasible to generate genome-edited rats by delivery of CRISPR/Cas9 components, the tyrosinase (Tyr) gene was used as a target. Some pups showed albino-colored coat, indicating disruption of wild-type Tyr gene allele. Furthermore, we confirm that rGONAD method can be used to introduce genetic changes in rat genome by the ssODN-based knock-in. ConclusionsWe first establish the rGONAD method for generating genome-edited rats. We demonstrate high efficiency of the rGONAD method to produce knock-out and knock-in rats, which will facilitate the production of rat genome engineering experiment. The rGONAD method can also be readily applicable in mammals such as guinea pig, hamster, cow, pig, and other mammals.
See https://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-018-0430-5
Figure 1: Determination of optimal electroporation efficiency for rGONAD. a Scheme of experimental procedures for evaluation of electroporation efficiency using GONAD method. b SZX7 stereomicroscope and Super Electroporator NEPA21. c Tetramethylrhodamine-labelled dextran is instilled into the oviduct lumen using a micropipette inserted through the oviductal wall near the infundibulum. d After the injection, the oviductal regions were covered with a piece of wet paper, and then, electroporation is performed using tweezer-type electrodes. e, f Scheme (e) or diagram (f) of electroporation conditions delivered of by an electroporator. This electroporation parameters were; Poring pulse; 40 V, 5 msec pulse, 50 msec pulse interval, number of pulse 3 times, 10% decay (± pulse orientation) and Transfer pulse; 10 V, 50 msec pulse, 50 msec pulse, number of pulse 6 times, 40% decay (± pulse orientation). g, h Fluorescence analysis of Tetramethylrhodamine-labelled dextran using GONAD method. i-k Graph shows analysis of the percentage of electroporation efficiency in WKY (i), DA (j), and DA x WKY (k). Ova, ovary; Ovi, Oviduct; Ute, uterus. Scale bars: 50 μm (g, h) |
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