Targeted gene delivery in the cricket brain, using in vivo electroporation

Matsumoto, Chihiro Sato; Shidara, Hisashi; Matsuda, Kōji; Nakamura, Taro; Mito, Taro; Matsumoto, Y.; Oka, Kotaro; Ogawa, Hiroto

doi:10.1016/j.jinsphys.2013.10.001

Cited by 6 publications

(5 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Specifically, following intense microinjection and electroporation treatments, high survival rates were observed for both experimental rounds. These mortality results are in agreement with a number of studies where microinjection and electroporation treatments were conducted on insects with survival rates of 60% or higher ( Matsumoto et al 2013 , Ando and Fujiwara 2013 ).…”

Section: Discussionsupporting

confidence: 91%

“…1b ). Two platinum probes covered with PBS buffer were pressed to either side of the aphid, similar to Matsumoto et al (2013) . Five pulses (approximately 3 s each) at 5 V were delivered to the aphid to promote sgRNA/Cas9 gene editing.…”

Section: Methodsmentioning

confidence: 99%

“…In vivo electroporation has been used in a wide range of organisms and involves applying electric shocks to the study subject to promote introduction of molecular agents ( Sugimoto and Tsuchida 2015 ). Recently, Matsumoto et al (2013) successfully applied in vivo electroporation to perform targeted gene delivery in the cricket brain. This method included five electrical pulses following microinjection and resulted in a high efficiency of plasmid transfer.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Efficacy of In Vivo Electroporation on the Delivery of Molecular Agents into Aphid (Hemiptera: Aphididae) Ovarioles

Jamison

Thairu

Hansen

2018

Journal of Insect Science

View full text Add to dashboard Cite

While the wealth of genomic data presently available is increasing rapidly, the advancement of functional genomics technologies for the large majority of these organisms has lagged behind. The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 system is an emerging gene-editing technology derived from a bacterial adaptive immune system that has proven highly effective in multiple model systems. Here, the CRISPR/Cas9 system was delivered into the ovarioles of the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphididae), with a new delivery method utilizing in vivo electroporation. To validate gene-editing, a target sequence within the marker tor pigment gene was chosen, and gene-editing was predicted to result in white pigmentation in the offspring of treated adult aphids. Adult aphids (10-d old) were injected with the tor single guide RNA and Cas9 complex and subsequently subjected to electroporation. Adult aphids were given 4 d to produce viviparous offspring. After offspring developed for 6 d, DNA was extracted and sequenced to validate if CRISPR/Cas9-directed gene editing occurred. A survival rate over 70% was found in treated adult aphids. A distinct white pigmentation was found in 2.5% of aphids; however, gene-editing within the target sequence was not found in any of the individuals screened. Presence of white aphids without gene-editing suggests other mechanisms may have influenced pigmentation. High survival rates in experimental treatments demonstrate the robustness of this new technique, and further refinement of this technique may prove it as an effective functional genomics tool for viviparous insects and/or gene editing at a somatic level.

show abstract

Section: Discussionsupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Efficacy of In Vivo Electroporation on the Delivery of Molecular Agents into Aphid (Hemiptera: Aphididae) Ovarioles

Jamison

Thairu

Hansen

2018

Journal of Insect Science

View full text Add to dashboard Cite

show abstract

“…Procedure of the dye loading was similar to the method for microinjection of DNA plasmid for electroporation in our previous work [17]. After crickets were anesthetized by cooling on crushed ice, the wings and legs were removed and the body was pinned dorsal side up on a silicone platform.…”

Section: Dye Loadingmentioning

confidence: 99%

Ca2+ imaging of cricket protocerebrum responses to air current stimulation

Ogawa

Kajita

2015

Neuroscience Letters

Self Cite

View full text Add to dashboard Cite

Crickets (Gryllus bimaculatus) use the cercal sensory system at the rear of the abdomen to detect air currents and direct predator avoidance behavior. Sensory information regarding the direction and dynamic properties of air currents is processed within the terminal abdominal ganglion, and conveyed by ascending giant interneurons (GIs) to higher centers including the brain. However, the brain region responsible for decoding cercal sensory information has not yet been identified, nor the response properties within the brain characterized. In this study, we performed in vivo Ca 2+ imaging to investigate wind-evoked neural activities within the cricket protocerebrum. Ca 2+ responses to air current stimuli were observed at peripheral regions of the ventrolateral neuropile (VLNP) where projection of GIs' axon terminals has been observed in larvae. The wind-evoked Ca 2+ response had temporal dynamics and directional sensitivity that varied with different recorded regions displaying transient or sustained Ca 2+ increases. Individual cells showed Ca 2+ elevation in response to air currents from a specific angle, while stimuli from a different angle evoked decreased signals. Removing the antennae reduced the air-current-evoked responses in VLNP, suggesting contribution of sensory inputs from antennae in addition to the cercal inputs. The VLNP is presumably an integrative center for mechanosensory processing from antennae and cerci where directional information is primarily decoded by protocerebral 3 neurons.

show abstract

“…Plasmid DNA harboring an enhanced green fluorescent protein (EGFP) gene was injected into adult cricket brains, followed by electroporation at a sufficient voltage. Expression of EGFP was observed within the brain tissue [Matsumoto et al, 2013]. Gene therapies for cancer utilizing in vivo electroporation have been proved effective in a number of experimental murine tumor models.…”

Section: Dna Deliverymentioning

confidence: 99%