Single Neuron Recording: Progress Towards High-Throughput Analysis

Alegria, Andrew D; Joshi, Amey; OBrien, Jacob; Kodandaramaiah, Suhasa B.

doi:10.2217/bem-2020-0011

Cited by 5 publications

(4 citation statements)

References 22 publications

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“…2012 ; 2013 ; 2016 , 2018 , 2022 ; Wu et al 2016 ; Annecchino et al 2017 ; Suk et al 2017 ; Holst et al 2019 ; Kolb et al . 2019 ; Alegria et al . 2020 ; Joshi et al .…”

Section: Discussionunclassified

High-throughput genetic manipulation of multicellular organisms using a machine-vision guided embryonic microinjection robot

Alegria,

Joshi,

Mendana

et al. 2024

Genetics

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Microinjection is a technique used for transgenesis, mutagenesis, cell labeling, cryopreservation, and in vitro fertilization in multiple single and multicellular organisms. Microinjection requires specialized skills and involves rate limiting and labor-intensive preparatory steps. Here we constructed a machine vision guided generalized robot that fully automates the process of microinjection in fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio) embryos. The robot uses machine learning models trained to detect embryos in images of agar plates and identify specific anatomical locations within each embryo in 3D space using dual view microscopes. The robot then serially performs a microinjection in each detected embryo. We constructed and used three such robots to automatically microinject tens of thousands of Drosophila and zebrafish embryos. We systematically optimized robotic microinjection for each species and performed routine transgenesis with proficiency comparable to highly skilled human practitioners while achieving up to 4x increases in microinjection throughput in Drosophila. The robot was utilized to microinject pools of over 20,000 uniquely barcoded plasmids into 1,713 embryos in two days to rapidly generate more than 400 unique transgenic Drosophila lines. This experiment enabled a novel measurement of the number of independent germline integration events per successfully injected embryo. Finally, we showed that robotic microinjection of cryoprotective agents in zebrafish embryos significantly improves vitrification rates and survival of cryopreserved embryos post-thaw as compared to manual microinjection. We anticipate that the robot can be used to carry out microinjection for genome-wide manipulation and cryopreservation at scale in a wide range of organisms.

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“…2012 ; 2013 ; 2016 , 2018 , 2022 ; Wu et al 2016 ; Annecchino et al 2017 ; Suk et al 2017 ; Holst et al 2019 ; Kolb et al . 2019 ; Alegria et al . 2020 ; Joshi et al .…”

Section: Discussionunclassified

High-throughput genetic manipulation of multicellular organisms using a machine-vision guided embryonic microinjection robot

Alegria,

Joshi,

Mendana

et al. 2024

Genetics

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“…Recordings were comparable in quality and stability in terms of resting membrane potential, input resistance, holding duration and spike magnitude, to manual in vivo studies (Margrie et al, 2002;DeWeese, 2007) and "blind" robotic systems (Kodandaramaiah et al, 2012(Kodandaramaiah et al, , 2018, across a range of recording depths. For more information on the state-of-the-art in autonomous patch-clamping, consult the reviews by Annecchino and Schultz (2018), Suk et al (2019) and Alegria et al (2020).…”

Section: 3mentioning

confidence: 99%

Robotic automation of “blind” and two-photon targeted patch-clamp electrophysiology in vivo

Gonzalez¹,

Kgwarae²,

Annecchino³

et al. 2021

Preprint

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“…First, ion channels are typically expressed in mammalian cells, which can be slow and expensive to maintain and scale. Second, the measurement of ion channel activity is difficult to parallelize, especially since it may be necessary to measure ion channel activity in real time ( 5 , 6 ). Finally, the rational design of ion channels is often nontrivial since the relationship between ligand binding and pore opening is often mediated through complex allosteric interactions ( 7 ).…”

mentioning

confidence: 99%

“…One particularly attractive candidate ligand-gated ion channel for yeast expression is the P2X family of purinergic ion channels, which consists of P2X subtypes 1 to 7 ( 15 ) These receptors are involved in nociception ( 16 ), immunomodulation ( 17 , 18 ), taste ( 19 , 20 ), and hearing ( 21 ). P2X has been linked to deafness ( 5 ), cardiac dysfunction ( 22 ), and cancer ( 23 ). For the purposes of engineering ion channels in yeast, these receptors are promising candidates for recombinant expression because (a) most of the subtypes form a homotrimer, which simplifies channel expression and assembly and (b) they are cation-nonselective and can thus be assayed via common calcium indicators.…”

mentioning

confidence: 99%

Engineering a human P2X2 receptor with altered ligand selectivity in yeast

Gardner,

Tramont,

Bachanová

et al. 2024

Journal of Biological Chemistry

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Single Neuron Recording: Progress Towards High-Throughput Analysis

Cited by 5 publications

References 22 publications

High-throughput genetic manipulation of multicellular organisms using a machine-vision guided embryonic microinjection robot

High-throughput genetic manipulation of multicellular organisms using a machine-vision guided embryonic microinjection robot

Robotic automation of “blind” and two-photon targeted patch-clamp electrophysiology in vivo

Engineering a human P2X2 receptor with altered ligand selectivity in yeast

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