Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis.
Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.
Department of Evolution and Developmental Biology, Zoological Institute, University Innsbruck.
Willi
W
Kari
Department of Evolution and Developmental Biology, Zoological Institute, University Innsbruck.
Fan
F
Zeng
Department of Evolution and Developmental Biology, Zoological Institute, University Innsbruck.
Lena
L
Zitzelsberger
Department of Evolution and Developmental Biology, Zoological Institute, University Innsbruck.
Johannes
J
Will
Department of Evolution and Developmental Biology, Zoological Institute, University Innsbruck; Ute.Rothbaecher@uibk.ac.at.
Ute
U
Rothbächer
D000818:Animals; D002938:Ciona intestinalis / Q000166:cytology / Q000196:embryology / Q000235:genetics*; D018274:Electroporation / Q000379:methods*; D018014:Gene Transfer Techniques; D006801:Humans; D008845:Microinjections / Q000379:methods*