Premium
Parameters affecting efficiency of in ovo electroporation of the avian neural tube and crest
Author(s) -
Simkin Johanna E.,
Zhang Dongcheng,
Ighaniyan Samiramis,
Newgreen Donald F.
Publication year - 2014
Publication title -
developmental dynamics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.634
H-Index - 141
eISSN - 1097-0177
pISSN - 1058-8388
DOI - 10.1002/dvdy.24163
Subject(s) - electroporation , in ovo , biology , transfection , plasmid , quail , notochord , neural tube , neural crest , embryo , transposase , cranial neural crest , green fluorescent protein , chorioallantoic membrane , microbiology and biotechnology , anatomy , embryogenesis , genetics , mutant , cell culture , endocrinology , gene , transposable element
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. Developmental Dynamics 243:1440–1447, 2014 . © 2014 Wiley Periodicals, Inc.