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

Abstract Background Improved genome-editing via oviductal nucleic acids delivery (i-GONAD) is a new technology that facilitates in situ genome-editing of mammalian zygotes exiting the oviductal lumen. The i-GONAD technology has been developed for use in mice, rats, and hamsters; however, oligonucleotide (ODN)-based knock-in (KI) is more inefficient in rats than mice. To improve the efficiency of i-GONAD in rats we examined KI efficiency using three guide RNAs (gRNA), crRNA1, crRNA2 and crRNA3. These gRNAs recognize different portions of the target locus, but also overlap each other in the target locus. We also examined the effects of commercially available KI -enhancing drugs (including SCR7, L755,507, RS-1, and HDR enhancer) on i-GONAD-mediated KI efficiency. Results The KI efficiency in rat fetuses generated after i-GONAD with crRNA2 and single-stranded ODN was significantly higher (24%) than crRNA1 (5%; p < 0.05) or crRNA3 (0%; p < 0.01). The KI efficiency of i-GONAD with triple gRNAs was 11%. These findings suggest that KI efficiency largely depends on the type of gRNA used. Furthermore, the KI efficiency drugs, SCR7, L755,507 and HDR enhancer, all of which are known to enhance KI efficiency, increased KI efficiency using the i-GONAD with crRNA1 protocol. In contrast, only L755,507 (15 μM) increased KI efficiency using the i-GONAD with crRNA2 protocol. None of them were significantly different. Conclusions We attempted to improve the KI efficiency of i-GONAD in rats. We demonstrated that the choice of gRNA is important for determining KI efficiency and insertion and deletion rates. Some drugs (e.g. SCR7, L755,507 and HDR enhancer) that are known to increase KI efficiency in culture cells were found to be effective in i-GONAD in rats, but their effects were limited.

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SAT-111 Rat genome editing by rGONAD (rat Genome-editing via Oviductal Nucleic Acids Delivery) method

Kidney International Reports ◽

10.1016/j.ekir.2019.05.139 ◽

2019 ◽

Vol 4 (7) ◽

pp. S52

Author(s):

M. Matsuyama ◽

T. Koyano ◽

T. Kobayashi ◽

M. Namba ◽

M. Fukushima

Keyword(s):

Nucleic Acids ◽

Genome Editing ◽

Delivery Method ◽

Nucleic Acids Delivery ◽

Rat Genome

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Successful i-GONAD in Brown Norway Rats by Modification of in vivo Electroporation Conditions

10.21926/obm.genet.2004121 ◽

2020 ◽

Vol 4 (4) ◽

Author(s):

Shuji Takabayashi ◽

◽

Takuya Aoshima ◽

Yukari Kobayashi ◽

Hisayoshi Takagi ◽

...

Keyword(s):

Genome Editing ◽

Ex Vivo ◽

Preimplantation Embryos ◽

Brown Norway ◽

Sprague Dawley ◽

In Vivo Electroporation ◽

Nucleic Acids Delivery ◽

A Current

Improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) was developed for in situ genome editing of the preimplantation embryos present within the oviductal lumen of mice. This method is based on intra-oviductal instillation of genome editing components and subsequent in vivo electroporation (EP) in the entire oviduct. Therefore, i-GONAD differs from the previous methods (i.e., zygote microinjection and in vitro EP) in producing genome-edited mice, which relied on ex vivo handling of preimplantation embryos and egg transfer to the recipient females. We have previously demonstrated that i-GONAD can be successfully applied to produce genome-edited rats, including albino Sprague-Dawley and albino Lewis rats (however, not pigmented Brown Norway [BN] rats). We observed that the successful i-GONAD was dependent on the mouse strain used; for example, in random-bred mice, such as ICR and C3H/He × C57BL/6, it was successful under relatively stringent electrical conditions but not in the C57BL/6 strain. Under less stringent conditions, i-GONAD was successful in the C57BL/6 strain. We speculated that this would also be true for i-GONAD using BN rats. On applying a current of >500 mA, we failed to obtain rat offspring (fetuses/newborns); however, i-GONAD under a current of 100-300 mA using NEPA21 (NEPA GENE) led to the production of genome-edited BN rats with efficiencies of 75%-100%. Similarly, i-GONAD, under a current of 150-200 mA using CUY21EDIT II (BEX Co.) led to the production of genome-edited BN rats with efficiencies of 24%-55%. These experiments suggest the importance of selecting the appropriate current value, depending on the rat strain used, when performing i-GONAD.

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Cationic gas-filled microbubbles for ultrasound-based nucleic acids delivery

Bioscience Reports ◽

10.1042/bsr20160619 ◽

2017 ◽

Vol 37 (6) ◽

Cited By ~ 11

Author(s):

Anthony Delalande ◽

Colette Bastié ◽

Lucie Pigeon ◽

Simona Manta ◽

Matthias Lebertre ◽

...

Keyword(s):

Nucleic Acids ◽

Electrostatic Interactions ◽

Low Frequency ◽

Target Tissue ◽

High Concentration ◽

Non Invasive ◽

Efficient Delivery ◽

Low Frequency Ultrasound ◽

Nucleic Acids Delivery

The use of ultrasound has gained great interest for nucleic acids delivery. Ultrasound can reach deep tissues in non-invasive manner. The process of sonoporation is based on the use of low-frequency ultrasound combined with gas-filled microbubbles (MBs) allowing an improved delivery of molecules including nucleic acids in the insonified tissue. For in vivo gene transfer, the engineering of cationic MBs is essential for creating strong electrostatic interactions between MBs and nucleic acids leading to their protection against nucleases degradation and high concentration within the target tissue. Cationic MBs must be stable enough to withstand nucleic acids interaction, have a good size distribution for in vivo administration, and enough acoustic activity to be detected by echography. This review aims to summarize the basic principles of ultrasound-based delivery and new knowledge acquired in these recent years about this method. A focus is made on gene delivery by discussing reported studies made with cationic MBs including ours. They have the ability for efficient delivery of plasmid DNA (pDNA), mRNA or siRNA. Last, we discuss about the key challenges that have to be faced for a fine use of this delivery system.

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