title: Parameters affecting efficiency of in ovo electroporation of the avian neural tube and crest.
abstract: BACKGROUND
  Many variations in avian in ovo transfection of the neural tube/crest have been reported, but never compared quantitatively.
  
  
  RESULTS
  Genome integrating pT2K-CAGGS-GFP and pCAGGS-T2TP transposase plasmids were co-electroporated into quail E2 embryo trunk neural tube and the proportion of GFP-expressing neural cells was counted 1 and 7 days later. Electroporation efficiency increased with plasmid concentration and pulse number but plateaued at, respectively, above 1.25 µg/µL and 3 pulses. Bilateral electroporation transfected more cells than unilateral but less than that anticipated by doubling the unilateral treatment. Holding the concentration of GFP plasmid constant and varying the transposase plasmid concentration revealed an optimum ratio of, in this case, 4:1 (1.2 µg/µL:0.3 µg/µL). Leaving transfected embryos to E9 confirmed that expression was maintained in vivo with the transposase system, but declined with non-integrated plasmid. Transfection of neural crest cells was low if electroporated less than 6-8 hr before emigration. We propose this indicates loss of epithelial integrity well prior to exit. We suggest this event be termed epithelio-mesenchymal transition sensu stricto, whereas the term delamination be reserved for the later emigration from the neural epithelium.
  
  
  CONCLUSIONS
  Co-electroporation in ovo must take into account plasmid(s) concentration and ratio, pulse number, pulse directionality, and timing.
authors.affiliation: Embryology Laboratory, Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, VIC, Australia.
authors.forename: Johanna E
authors.initials: JE
authors.lastname: Simkin
authors.forename: Dongcheng
authors.initials: D
authors.lastname: Zhang
authors.forename: Samiramis
authors.initials: S
authors.lastname: Ighaniyan
authors.forename: Donald F
authors.initials: DF
authors.lastname: Newgreen
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002452:Cell Count; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods* / Q000592:standards; D049452:Green Fluorescent Proteins / Q000378:metabolism; D009432:Neural Crest / Q000378:metabolism*; D054259:Neural Tube / Q000166:cytology / Q000378:metabolism*; D011784:Quail / Q000196:embryology*; D014162:Transfection / Q000379:methods*; D019895:Transposases / Q000378:metabolism; D015053:Zygote / Q000378:metabolism*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019895:Transposases
keywords: cell counts; delamination; epithelium-mesenchyme transition; neural epithelium; plasmid; transposon