Transfection into Mouse/Rat Zygotes in Oviducts by Electroporation (i-GONAD)

NEPA21

Tweezer Electrodes

Cables, Foot Switch & Others

Transfection into Mouse/Rat Zygotes in Oviducts by Electroporation (i-GONAD)

APPLICATIONS

Generation of genome-edited rats by the rGONAD method

Optimization of conditions for electroporation in the rGONAD method

  1. Experimental procedure for evaluating electroporation efficiency in the GONAD method
  2. Stereo microscope SZX7 (Olympus) and super electroporator NEPA21 (Nepa Gene Co., Ltd.)
  3. Inject Tetramethylrhodamine-labeled dextran into the oviductal lumen through the wall of the oviduct near the tubal fimbriae using a micropipette.
  4. After injection, cover the fallopian tube region with a Kimwipe soaked in PBS and cut to the appropriate size and electroporate using CUY652P2.5X4 (Tweezers w/2.5mm x 4mm concave platinum electrodes, Nepa gene Co., Ltd.) Ova: ovary, Ovi: oviduct, Ute: uterus
  5. Pulse waveform of electroporation
  6. NEPA21 electrical conditions
    • Poring Pulse Voltage: 40V, Pulse width: 5msec, Pulse interval: 50msec, Number of pulse: 3 times, Decay rate: 10%, Polarity: +/- (with switching)
    • Transfer Pulse Voltage: 10V, Pulse Width: 50msec, Pulse Interval: 50msec, Number of pulse: 6 times, Decay rate: 40%, Polarity: +/- (with switching)
  1. h. Fluorescence analysis of tetramethylrhodamine-labeled dextran Scale bar: 50 µm
  2. Fluorescence analysis graph of electroporation efficiency in rat WKY
  3. Fluorescence analysis graph of electroporation efficiency in rat DA
  4. Fluorescence analysis graph of electroporation efficiency in rat DA×WKY

Generation of Try gene knockout (KO) rats by rGONAD method

  1. Scheme of allele-specific genome editing for knock-out rats
    • The rGONAD method was performed in 0.75 day pregnant albino WKY crossed to agouti DA strains.
    • The fertilized eggs are (WKY x DA) F1 hybrid. The target sequence and PAM at Tyr locus are shown.
  2.  Some the editing rats had albino colored coats. 
  3. Direct sequencing results of wild-type F1 (upper; WT) or the editing (below; indel) rats. Red arrow indicates indel mutaion. 
  4. Sequence analysis of the pups showed a variety of indel mutation at the Tyr locus, as shown in red. 
  5. Graph shows analysis of the percentage of genome edited efficiency in Tyr gene in WKY female x DA male
  6. Graph shows analysis of the percentage of genome edited efficiency in Tyr gene in WKY female x male

Recovery of coat-color mutation in albino WKY rats using KI approach

  1. Scheme of hair color mutation repair
    • The rGONAD method was performed on WKY female rats at 0.75 days gestation mated with WKY male rats.
    • Diagram of target sequences, PAM sequences, and ssODNs at the Tyr locus.
  2. Non-agouti, hooded-spotted albino KI rats.

Mutation of F1 (WKY x DA) rats via Tyr

 

Courtesy of Dr. Makoto Matsuyama, Division of Molecular Genetics, Shigei Medical Research Institute
Kobayashi et al., BMC Biotechnology (2018)

PUBLICATIONS

  • KO_mice

Loss of Atg2b and Gskip Impairs the Maintenance of the Hematopoietic Stem Cell Pool Size

Shun-Suke Sakai, Atsushi Hasegawa, Ryosuke Ishimura, Naoki Tamura, Shun Kageyama, Satoko Komatsu-Hirota, Manabu Abe, Yiwei Ling, Shujiro Okuda, Manabu Funayama, Mika Kikkawa, Yoshiki Miura, Kenji Sakimura, Ichiei Narita, Satoshi Waguri, Ritsuko Shimizu, Masaaki Komatsu

Mol Cell Biol. 2022 Jan 20;42(1):e0002421.

  • KO_rats

Creation of X-linked Alport Syndrome Rat Model with Col4a5 Deficiency

Masumi Namba, Tomoe Kobayashi, Mayumi Kohno, Takayuki Koyano, Takuo Hirose, Masaki Fukushima, Makoto Matsuyama

Sci Rep. 2021 Oct 21;11(1):20836

  • KO_mice
  • KI_mice
  • KO_rats
  • KI_rats

GONAD: A new method for germline genome editing in mice and rats

Masumi Namba, Tomoe Kobayashi, Takayuki Koyano, Mayumi Kohno, Masato Ohtsuka, Makoto Matsuyama

Dev Growth Differ. 2021 Aug 25.

  • KI_mice

Functional validation of epitope-tagged ATF5 knock-in mice generated by improved genome editing of oviductal nucleic acid delivery (i-GONAD)

Haruo Nakano, Shiori Kawai, Yusaku Ooki, Tomoki Chiba, Chiharu Ishii, Takumi Nozawa, Hisako Utsuki, Mariko Umemura, Shigeru Takahashi, Yuji Takahashi

Cell Tissue Res . 2021 Jul;385(1):239-249.

  • KO_mice

An efficient i-GONAD method for creating and maintaining lethal mutant mice using an inversion balancer identified from the C3H/HeJJcl strain

Satoru Iwata, Takahisa Sasaki, Miki Nagahara, Takashi Iwamoto

G3 Genes|Genomes|Genetics, jkab194, 03 June 2021

  • KO_mice

Improvement of genome editing by electroporation using embryos artificially removed cumulus cells in the oviducts

Takehito Kaneko, Shungo Tanaka

Biochem Biophys Res Commun. 2020 Jul 5;527(4):1039-1042.

  • KO_mice

In utero gene transfer system for embryos before neural tube closure reveals a role for Hmga2 in the onset of neurogenesis

Kuwayama N, Kishi Y, Maeda Y, Nishiumi Y, Suzuki Y, Koseki H, Hirabayashi Y, Gotoh Y.

bioRxiv May 15, 2020

  • KI_mice

Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice

Masahiro Sato, Rico Miyagasako, Shuji Takabayashi, Masato Ohtsuka, Izuho Hatada, Takuro Horii

Cells, 9 (3) 2020 Feb 26

  • KO_mice

Simple and large-scale chromosomal engineering of mouse zygotes via in vitro and in vivo electroporation.

Iwata S, Nakadai H, Fukushi D, Jose M, Nagahara M, Iwamoto T

Sci Rep. 2019 Oct 11;9(1):14713.

  • KO_mice

The p21 dependent G2 arrest of the cell cycle in epithelial tubular cells links to the early stage of renal fibrosis.

Koyano T, Namba M, Kobayashi T, Nakakuni K, Nakano D, Fukushima M, Nishiyama A, Matsuyama M.

Sci Rep . 2019 Aug 19;9(1):12059.

  • KO_rats
  • KI_rats

i-GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats.

Takabayashi S, Aoshima T, Kabashima K, Aoto K, Ohtsuka M, Sato M

Sci Rep. 2018 Aug 13;8(1):12059.

  • KO_rats
  • KI_rats

Successful production of genome-edited rats by the rGONAD method.

Kobayashi T, Namba M, Koyano T, Fukushima M, Sato M, Ohtsuka M, Matsuyama M

BMC Biotechnol. 2018 Apr 2;18(1):19

Electroporation

Drug Delivery and Transfection

Electro Cell Fusion

Fluorescent Staining

Single-Cell/Micro-Particle Transfer

Cell Freezing

Mechanical Vibration