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High-throughput genetic manipulation of multicellular organisms using a machine-vision guided embryonic microinjection robot.
Alegria, Andrew D; Joshi, Amey S; Mendana, Jorge Blanco; Khosla, Kanav; Smith, Kieran T; Auch, Benjamin; Donovan, Margaret; Bischof, John; Gohl, Daryl M; Kodandaramaiah, Suhasa B.
Afiliación
  • Alegria AD; Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
  • Joshi AS; Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
  • Mendana JB; University of Minnesota Genomics Center, University of Minnesota, Minneapolis, MN 55455, USA.
  • Khosla K; Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
  • Smith KT; Department of Fisheries, Wildlife and Conservation Biology, University of Minnesota, St. Paul, MN 55108, USA.
  • Auch B; University of Minnesota Genomics Center, University of Minnesota, Minneapolis, MN 55455, USA.
  • Donovan M; University of Minnesota Genomics Center, University of Minnesota, Minneapolis, MN 55455, USA.
  • Bischof J; Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
  • Gohl DM; Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
  • Kodandaramaiah SB; University of Minnesota Genomics Center, University of Minnesota, Minneapolis, MN 55455, USA.
Genetics ; 226(4)2024 04 03.
Article en En | MEDLINE | ID: mdl-38373262
ABSTRACT
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 4× 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 2 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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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Robótica Límite: Animals / Humans Idioma: En Revista: Genetics Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Robótica Límite: Animals / Humans Idioma: En Revista: Genetics Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos