An automated system for intracellular and intranuclear injection.
The Xenopus oocyte expression system has played an important role in the study of cellular proteins, particularly in the field of membrane physiology; expression of transporters and ion channels has significantly advanced our knowledge of these membrane proteins and the rapid and easy expression of mutants has been crucial in many structure-function studies. Xenopus oocytes are an expression system in many ligand-binding assays and in functional screening for ion channel modulators. Several commercially available automated technologies use this system, generating a demand for large numbers of oocytes injected with ion channel genes. Injection of oocytes with genetic material is generally carried out manually. Here we describe an automated system capable of injecting up to 600 oocytes per hour. Oocytes are contained in microplates with conical wells, a simple calibration procedure by the operator is required and pipette filling and oocyte injection are carried out automatically. Following intracellular injection of mRNA coding for ligand-gated ion channels close to 100% of oocytes tested positive for expression, and intranuclear injection of cDNA gave a rate of expression >50%. Moreover, we demonstrate that this method can also be successfully applied to inject zebrafish embryos and could be extended to other cell types.
Hogg
Ron C
RC
Department of Neurosciences Fondamentales, Medical Faculty, University of Geneva, Switzerland. Ronald.Hogg@medecine.unige.ch
Bandelier
Florence
F
Benoit
Audrey
A
Dosch
Roland
R
Bertrand
Daniel
D
D000818:Animals; D001331:Automation / Q000295:instrumentation / Q000379:methods*; D001681:Biological Assay / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics / Q000648:ultrastructure; D003584:Cytological Techniques / Q000295:instrumentation / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005260:Female; D007473:Ion Channels / Q000187:drug effects / Q000235:genetics; D008845:Microinjections / Q000295:instrumentation / Q000379:methods*; D009865:Oocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000494:pharmacology*; D014982:Xenopus laevis; D015027:Zebrafish
D018076:DNA, Complementary; D007473:Ion Channels; D012333:RNA, Messenger