Animal Cell Culture, rGONAD Method,Gene Editing,Summary of Successful production of genome-edited rats by the rGONAD method. By:- Tomoe Kobayashi, Masumi Namba, Takayuki Koyano, Masaki Fukushima, Masahiro Sato, Masato Ohtsuka and Makoto Matsuyama Kobayashi et al. BMC Biotechnology (2018) 18:19 https://doi.org/10.1186/s12896-018-0430-5

1. Genome Edited Rats
Submitted By:-
Ilika Kaushik
M.Tech Biotechnology
19PBT004
1st Sem.
Submitted To:-
Dr. Bhaswati Banerjee
Ma’am
Asst. Professor
SoBT, GBU.

2. Topic:-
Successful production of genome-edited rats by the
rGONAD method.
By:-
Tomoe Kobayashi, Masumi Namba, Takayuki
Koyano, Masaki Fukushima, Masahiro Sato, Masato
Ohtsuka and Makoto Matsuyama
Kobayashi et al. BMC Biotechnology (2018) 18:19
https://doi.org/10.1186/s12896-018-0430-5

3. Index
S.No. Contents
1. Introduction
2. Background
3. Procedure
4. Results
5. Discussion
6. Summary

4. Introduction
• Recent researches in CRISPR/Cas9 has made it a very
efficient gene editing tool in various organisms.
• Genome editing via Oviductal Nucleic Acid Delivery
(GONAD) is a novel method developed in mice.
• A novel, in vivo genome editing technique.
• This editing system doesn’t require in vivo handling of
embryos.

5. Background
• The laboratory rat (Rattus norvegicus) is used as a model for
various experiments.
• Generally used in many studies to model a specified trait of
human diseases and for testing the drugs.
• Despite its usefulness production of genetically engineered
rats has yet not been extensively proceeded during the past
decades.
• Development of successful rat ES cell culture was first made
in 2010.

6. • In 2011 to 2013, a more convenient and simpler technology,
called genome-editing technology as exemplified by zinc
finger nucleases (ZFN) transcription activator-like effector
nucleases and CRISPR/Cas9-capable of modifying a specific
gene function in mammals came in use.
• These technologies, helped to create genetically modified
rats in a more convenient manner than ever.

7. Procedure
Collection of
specimen
(animals-rats).
Preparation of
fluorescence-
labelled
dextran.
Preparation of
CRISPR/Cas9
reagents.
rGONAD
Method.
Genotyping
Fluorescent
Siganal
Detection.

9. Results

10. 1. Determination of optimal electroporation
efficiency for rGONAD.

11. 2. rGONAD-based gene disruption (KO).

12. 3. Recovery of coat-
color mutation using
ssODN-based knock-
in (KI) approach.

13. Discussion
• The present study showed that the most suitable conditions
for rGONAD method confirmed by electroporation
efficiency.
• The rGONAD method allows efficient production of gene
disrupted rats using CRISPR/Cas9 system.
• Furthermore, high efficiency of rGONAD method to produce
knock-out and knock-in rats is demonstrated.
• Recently, electroporation-mediated in vitro transfection of
isolated pre-implantation embryos has been reported in rats
(TAKE methods)
• The rGONAD method is more reliable that does not require
any specific skills and equipment such as micromanipulator
and inverted microscope.

14. • The rGONAD method does not require euthanasia of
pregnant females, unlike in the traditional approaches where
females need to be sacrificed for isolating zygotes for
introduction of genome editing components ex vivo.
• Moreover, it was founded that some female rats which
underwent rGONAD method were able to deliver the pups.
• This indicate that the operated oviducts in the treated rats
retained normal reproductive functions- pregnancy and
delivery.
• Therefore this method can be suggested for producing
genome editing animals that can be useful in variety of
experimentation.

15. Conclusion
• The rGONAD method will facilitate the production of rat
genome engineering experiments in many laboratories.
• Furthermore, rGONAD method can be readily applicable
where traditional gene targeting using ES cells will not be
well established in mammals such as guinea pig, hamster,
cow, pig, and other mammals.