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TECHNIQUES

Parameters Affecting Efficiency of In Ovo Electroporationof the Avian Neural Tube and CrestJohanna E. Simkin, Dongcheng Zhang, Samiramis Ighaniyan, and Donald F. Newgreen*

Embryology Laboratory, Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville VIC, Australia

Background: Many variations in avian in ovo transfection of the neural tube/crest have been reported, but never compared quanti-tatively. Results: Genome integrating pT2K-CAGGS-GFP and pCAGGS-T2TP transposase plasmids were co-electroporated into quailE2 embryo trunk neural tube and the proportion of GFP-expressing neural cells was counted 1 and 7 days later. Electroporationefficiency increased with plasmid concentration and pulse number but plateaued at, respectively, above 1.25 mg/mL 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 ratioof, in this case, 4:1 (1.2 mg/mL:0.3 mg/mL). Leaving transfected embryos to E9 confirmed that expression was maintained in vivowith the transposase system, but declined with non-integrated plasmid. Transfection of neural crest cells was low if electroporatedless than 6–8 hr before emigration. We propose this indicates loss of epithelial integrity well prior to exit. We suggest this eventbe termed epithelio-mesenchymal transition sensu stricto, whereas the term delamination be reserved for the later emigrationfrom 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. VC 2014 Wiley Periodicals, Inc.

Key words: plasmid; transposon; neural epithelium; delamination; epithelium-mesenchyme transition; cell counts

Submitted 5 May 2014; First Decision 25 June 2014; Accepted 30 June 2014; Published online 13 July 2014

Introduction

Techniques for direct genetic manipulation in living embryos,particularly avian embryos, include plasmids introduced by elec-troporation in vivo (Dickinson et al., 2002). Electroporation useselectric current pulses (usually square wave) to create poreswithin the cell membrane and simultaneously drive charged mac-romolecules into the cell (Phez et al., 2005); in the case of plas-mids, the direction is anode-ward. For in vivo electroporation (inavian embryos often termed in ovo electroporation), plasmid con-struct is injected locally and electrodes are placed either side ofthe region (Krull, 2004). Most commonly and effectively, theplasmid is injected into a tissue cavity bounded by an epitheliumsuch as the neural tube. Electroporation alone has little effect onendogenous gene expression (Farley et al., 2011), so this tech-nique identifies the response to the introduced genetic material.

The parameters of delivery influence the success of the trans-fection, and electroporation parameters for avian embryonicectoderm and neurectoderm using the common L-shaped electro-des have been presented (Endo, 2012; Muramatsu, 2000; Sauka-Spengler and Barembaum, 2008; Croteau and Kania, 2011; Krull,2004; Kadison and Krull, 2008). Electroporation employing elec-trodes of different geometry have also been described (Brown

et al., 2012; Voiculescu et al., 2008), and techniques for the morechallenging mesenchymal cells are also detailed (Momose et al.,1999; Oberg et al., 2002; Scaal et al., 2004).

The outcomes in these studies, sometimes termed electropora-tion efficiency, have generally been assessed grossly and superfi-cially, by in some cases listing the proportion of electroporatedembryos that survived, that were subjectively normal at the levelof external morphology, and that showed at least some markerconstruct (usually GFP) expression. A few reports also assessedmicroscopic morphology and expression of specific molecules inthe transfected tissues. However, a drawback in assessing variouselectroporation parameters from these publications is that com-parisons often need to be drawn from different sources, not fromside-by-side studies. In addition, specimen number was often notreported, and quantitative measures of “efficiency” were notused. In fact, it was usually difficult to gauge exactly what wasmeant by the term efficiency: for example, this term could referto intensity of label expression per cell or tissue, or proportion ofpotentially transfectable cells that were transfected.

Broadly, it is clear that neural epithelial cells of young avianembryos are transfected at electrical parameters too low for olderembryos and do not survive parameters suitable for olderembryos, and that transfection of mesenchyme cells requireshigher voltage and plasmid concentration for effectiveness.Moreover, transfection efficiency is increased by high voltage,which increases pore diameter/number (Nesin et al., 2011) and

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Grant sponsor: NHMRC; Grant number: 607379.J. E. Simkin, D. Zhang, and S. Ighaniyan made equal contributionsto the work.*Correspondence to: Don Newgreen, Embryology Laboratory, MurdochChildren’s Research Institute, Royal Children’s Hospital, Flemington Rd.,Parkville VIC 3052, Australia. E-mail: don.newgreen@mcri.edu.au

Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy.24163/abstractVC 2014 Wiley Periodicals, Inc.

DEVELOPMENTAL DYNAMICS 243:1440–1447, 2014DOI: 10.1002/DVDY.24163

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maximises construct flux but this increases specimen damageand death. Lesser distance between electrodes and longer pulseduration have similar results. Optimizing parameters involvesbalancing voltage, pulse duration, and electrode separation. Mul-tiple pulsing in in vitro electroporations is a useful way toincrease transfection while combating the restrictions of voltageand pulse length required for optimal cell survival (Mahmoodet al., 2008). Embryos have been electroporated with 2 (Momoseet al., 1999) to 10 pulses (Oberg et al., 2002) without these beingclosely compared to our knowledge. The interval between pulsesis often not mentioned but is frequently 1 sec although 100, 250,and 500 msec have been reported (Voiculescu et al., 2008; Brownet al., 2012; Endo, 2012).

A wide range of GFP plasmid concentrations (1–5 mg/mL) forneural tube transfections are commonly used while up to 7 mg/mLis suggested to transfect mesenchyme cells (Scaal et al., 2004).Transfection has been found to be poor at concentrations of 0.25mg/mL or less for GFP plasmids compared to 1 mg/mL, but interest-ingly lacZ plasmid expression is easily detectable at the lowerconcentration (Momose et al., 1999), suggesting that the detect-ability of various markers is of importance. Different plasmidtypes have different efficiencies (Kadison and Krull, 2008), butthe tacit impression is that transfection efficiency improves con-tinuously with plasmid concentration. However, the relationshipbetween concentration and efficiency has not been explored indetail and never quantified. Morever, in some cases, low transfec-tion efficiency may be desired (Binder et al., 2012).

Unilateral electroporation produces the often useful propertythat the cathodal side is electroporated but non-transfected, andso can act as an internal control. To increase the number of cellstransfected from that achievable by unilateral electroporation,embryos have been electroporated bilaterally. An equal numberof pulses bilaterally has been used, but in other cases the embryoshave been electroporated with 5 pulses on one side, then 1 on theother (Ahlstrom and Erickson, 2009). The reason for choosingthese pulse numbers and ratios was not clear. Bidirectional elec-troporation of cells in suspension has led to the proposal that thesecond-directed pulses may remove some of the construct deliv-ered with the first-direction pulse(s) (Phez et al., 2005). It is notknown if this phenomenon occurs in in ovo electroporations.

The plasmids transfected may be episomally transcribed ormay be designed to integrate into the host cell’s genome (Takaha-shi et al., 2008). The former is rapidly expressed but is diluted byproliferation, whereas the latter is retained through each divisioncycle allowing for permanent expression (Sato et al., 2007). Toachieve genomic integration, co-electroporation of a Tol2-flanked CAGGS-EGFP construct with an episomally expressedTol2 transposase plasmid has been used at a 1:1 ratio (Sato et al.,2007). It is not known if this is the optimum ratio of these twoplasmids for efficiency of transfection. The longevity of expres-sion of the episomal versus genome-integration plasmids hasbeen measured in vitro (Sato et al., 2007), but not quantified invivo.

In this report, we used a commonly used electrode geometryand pulse voltage for in ovo electroporation of the E2 quailembryo neural tube. We tested for the first time co-electroporatedgenome-integrating plasmids by counting transfected versusnon-transfected cells to assess transfection efficiency quantita-tively. We varied plasmid concentration, pulse number, unilateralversus bilateral electroporation, and ratio of co-electroporatedplasmids. We also tested the duration of expression in vivo of

genome-integrating versus non-integrating plasmids. In additionto these studies on neural epithelial cells, we extended this studyto measuring transfection of NC cells (Kadison and Krull, 2008), asub-section of the early neural epithelium that migrate away, toinvestigate the effect of timing of electroporation relative to tim-ing of migration on transfection efficiency of NC cells.

Results and Discussion

Electroporation Efficiency Plateaus at Low PlasmidConcentration

Under the same pulse conditions (3 x 25 volts, 50 msec, 1 secinterval, unilateral) for trunk neural tube electroporation, pT2K-CAGGS-GFP plasmid plus pCAGGS-T2TP plasmid at concentra-tion of (both) 1.25 mg/mL (N¼17) induced about 45% of the cellson the electroporated (anodal) side to express GFP (Fig. 1A).Because the cathode-facing side of the neural tube had virtuallyno GFP transfected cells (see Exp. Proc.), we counted the numberof GFP-labelled cells and divided by half the total number of cellsof the entire neural tube segment to arrive at the proportiontransfected on the anode side. Halving the concentration of bothplasmids (0.625 mg/mL; N¼14) reduced the transfection rate to23%. This difference was highly statistically significant(P<0.001). However, the advantage of increasing concentrationplateaued. No significant difference (P¼ 0.91) in transfectionrates was found between a concentration of 1.25 mg/mL (45%),and 2.5 mg/mL, which gave 44% (N¼17) of cells transfected.

The average proportion of cells transfected will vary with theindividual plasmid properties (size, charge density) (Sauka-Spen-gler and Barembaum, 2008), but we show here that a plateau isreached at a plasmid concentration lower than has been typicallyemployed.

Electroporation Efficiency Plateaus at Low PulseNumber

At the above conditions and plasmids at concentration of (both)1.25 mg/mL, more pulses produced greater transfection (Fig. 1B).One pulse induced GFP expression in an average of 33% of neu-roepithelial cells on the transfected side (N¼19), whereas threepulses, as mentioned, induced 45% (N¼17). The differencebetween these was highly statistically significant (P<0.001).However, this plateaued, because embryos receiving 6 pulses hadan average of 44% neural tube cells GFP positive on the trans-fected side (N¼16), the slight difference between 3 and 6 pulsesbeing not statistically significant (P¼ 0.78). This plateau mayreflect a reduction in construct available with each succeedingpulse.

Bilateral Electroporation Increases the Number of CellsTransfected But Electroporation Efficiency Is Reduced

Bilaterally electroporated embryos (1.25 mg/mL; ratio 1:1; Fig. 1B,C) receiving 6 pulses in total involving both sides either as seriesof 3þ3 (N¼10), 1þ5 (N¼16), or 5þ1 (N¼15) pulses, had signifi-cantly more neuroepithelial cells in total transfected (30, 28,32%, respectively) than did embryos with 6 pulses to one side(6þ0, N¼16; counting both sides: 22%; Fig. 1C). However, wheneach side was counted individually, bilateral electroporation effi-ciency per side was always lower than that achieved by a similar

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number of pulses delivered unilaterally (Fig. 1B). In bilaterallyelectroporated embryos, the number of cells transfected wasbiased to the side undergoing the most pulses (Fig. 1D), and itappeared that the second side had an additional bias when equalpulses were applied to both sides (Fig. 1D). However, equal pulsenumbers to each side gave more equal transfection (absolute dif-

ference between two sides 5% for 3þ3) than strongly biasedbilaterally electroporation (absolute difference between two sides12 and 10% for 1þ5 and 5þ1, respectively, P<0.05) (Fig. 1E).

Phez et al. (2005) have suggested that there are two states ofplasmid interaction during electroporation: a DNA interactionand a DNA insertion state, the former but not the latter beingreversible by a reverse-directed electroporation. This was derivedfrom electroporation of cells in suspension, but it may also applyto in ovo electroporation. Our results are consistent with thenotion that the first pulse(s) caused less construct to be availablefor the second or reversed electroporation, whereas the reversedpulses meant less than expected transfection is retained in thepreviously electroporated side. However, some of the constructdelivered to the first side was stable since even a greater numberof reversed pulses (i.e., 1þ5) failed to remove it. This is consistentwith the notions of Phez et al. (2005) that there are both reversi-ble and irreversible states achieved after electroporation.

Transfection Efficiency of Genome-Integrating EGFPPlasmid Varies With Ratio of Transposase Plasmid

Employing a constant pT2K-CAGGS-GFP plasmid concentration(1.2 mg/mL), the amount of tol2 transposase plasmid pCAGGS-T2TP was varied (Fig. 2A). (Note: the experiments in this sectionand the next section were performed with 0.5-mm-diameter cir-cular electrodes.) Using equal quantities of both plasmids (1:1ratio), unilateral electroporation of neural tube yielded a transfec-tion rate of 24% on the transfected side (N¼50), while halvingthe amount of tol2 plasmid increased the transfection rate to 30%(N¼25) (P<0.05;1:1/2 relative to 1:1). At a 1:1/4 ratio, transfec-tion was similarly raised (32%, N¼25) (P<0.01; 1:1/4 relative to1:1). Reducing the amount of tol2 plasmid to 1/8th of the GFPplasmid lowered the transfection efficiency of the transfectedside of the neural tube to 16% (N¼26) (P<0.05 relative to 1:1).Worthy of note was the expression of GFP signal even with notol2 transposase plasmid (10%, N¼22; 1:0 ratio). Translation ofGFP in the absence of the tol2 transposase was also observed bySato and colleagues in an in vitro system using DF1 chicken cells,indicative of GFP being transcribed episomally from the pT2K-CAGGS-GFP plasmid (Sato et al., 2007).

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Fig. 1. The effects of plasmid concentration, electroporation pulsenumber, and administration methods on the proportion of transfectedneural tube cells. A: The proportion of transfected cells was signifi-cantly higher at 1.25 and 2.5 mg/mL plasmids concentrations comparedto 0.625 mg/mL (***P<0.001), but with no difference between 1.25 and2.5 mg/mL. B: The proportion of transfected cells was significantlyhigher with 3 and 6 pulses compared to 1 pulse (**P <0.01, ***P<0.001) but 3 and 6 pulses were not significantly different. Under thesame pulses conditions, the proportion of transfected cells on eachside of bilateral electroporations was significantly lower than the equiv-alent side of unilateral electroporations (yyP <0.01, yyyP <0.001 vs. 3þ0;zz P <0.01 vs. 1þ0; # P <0.05, ## P <0.01 vs. 3þ0 and 6þ0). C: Thepercentage of transfected cells, summing both sides of the neuraltube, was increased by bilateral electroporation (3þ3, 1þ5, and 5þ1groups) compared to unilateral (6þ0) group (*P <0.05; ** P <0.01).D: Each diamond represents the difference between the second sideand first side from each bilaterally electroporated neural tube. There isa slight transfection bias towards the second side electroporated. Barsare mean and 95% CI. E: In terms of the proportion of transfectedcells, absolute differences between the two sides of the NT were signif-icant higher in 1þ5 and 5þ1 groups compared to the 3þ3 group(*P <0.05).

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GFP Signal Persists In Vivo With Transposon/Transposase Plasmid Electroporation

Sato et al. (2007) reported GFP expression in DF1 cells in cell cul-ture with and without co-electroporation of the transposase plas-mid but, later, GFP expression was observed only in the co-transfected cells. This was consistent with these having the GFPplasmid integrated into their genome, and in the absence of tol2transposase, the GFP plasmid being diluted over time throughcellular proliferation. To verify that this occurred in ovo, wequantified GFP expression efficiency in unilateral electroporationof E2 quail embryos following their co-transfection with pT2K-CAGGS-GFP plus pCAGGS-T2TP plasmids or with pT2K-CAGGS-GFP only, extending incubation until E9 (Fig. 2B). Co-electroporation of pT2K-CAGGS-GFP and pCAGGS-T2TP plas-mids yielded a transfection efficiency after an additional 7 daysin vivo of 23% (N¼12), which was no different from 1 day afterelectroporation (24%). In contrast, when pT2K-CAGGS-GFP plas-mid was electroporated alone, transfection efficiency signifi-cantly declined from 10% at 1 day to 4% at 7 days (N¼14)(P<0.05).

This stability of expression from 1 to 7 days post-electroporation for the co-electroporated GFP plasmid comparedwith the decline for the single plasmid is consistent with episomalexpression of the pT2K-CAGGS-GFP plasmid being diluted in thedividing cells of the developing embryo. In this case in the neuraltube, many of the remaining GFP cells are likely to be non-dividing, terminally differentiated neurons.

Transfection of Early Migrating NC Cells RequiresElectroporation Many Hours Before Onset of Migration

NC cells are a frequent target for electroporation (Yokota et al.,2011; Kadison and Krull, 2008). Trunk neural tube was bilaterallyelectroporated (3þ3) at two ages termed “young” and “old” (seeExp. Proc.) and then neural tube was obtained from the sameaxial level. Overt NC cell emigration had not commenced at bothstages, but the two stages differed in the time gap before theexpected onset of NC migration, which was estimated by refer-ence to the avian NC migration timetable (see fig. 3 in Newgreenand Erickson, 1986). The approximate timing gap before NC emi-

gration at the rostral end was about 8 hr (for young) and 0–1 hr(for old). These neural tubes were established in in vitro NC cellmigration assays (Newgreen and Murphy, 2000). OutgrowingSox-E-positive NC cells included GFP-expressing and non-expressing cells. The distribution of GFP-labelled migrating NCcells showed different patterns depending on the stage and posi-tion of origin.

At caudal ends of both young (Fig. 3A; N¼11) and old (Fig.3B; N¼10) stage explants, cell counts indicated that about 30–36% of migrating cells expressed GFP at all regions of NC out-growth from proximal to distal (zone I to IV; Fig. 3E). The propor-tion of NC cells labelled was consistent with the proportion ofneural tube cells transfected under these electroporation condi-tions (see 3þ3, Fig. 1C). At the rostral ends (Fig. 3C, D), the pro-portion of transfected NC cells was similar to that of the caudalends in the proximal outgrowth zone (i.e., zone I) in both old andyoung explants (both 38%). In contrast, the proportion of trans-fected NC cells gradually fell to 13% (young) and 11% (old) at themost distal (zone IV) outgrowth zone. This decline was evidentmore proximally (zone III) in the old explants (14%) compared tothe young explants (21%) (P<0.05).

In these cultures, the NC cells that commence migration earliesttend to become distributed most distantly (Newgreen et al.,1979); therefore, we conclude that electroporation was least effi-cient at transfecting those NC cells that commenced migration ataxial levels closest in time to the nominal time of onset of NCmigration. This finding has great practical importance for electro-poration studies of the NC, as early migrating NC cells give riseto different ranges of derivatives to those commencing migrationlater (Weston and Butler, 1966), and may differ in specificationfrom the later migrating cells (Reedy et al., 1998; Harris andErickson, 2007; Krispin et al., 2010; Nitzan et al., 2013).

Redefining the Terms EMT and Delamination in theNeural Crest

Electroporation of the neural tube will obviously fail to transfectmesenchymal NC cells that have already exited the neural tube.At the 20-somite stage, the NC cells at and caudal to the last 2somites are all pre-migratory in the sense that none have yet leftthe confines of the neural tube (Newgreen and Erickson, 1986),

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Fig. 2. The effect of plasmid concentration ratios on the proportion of transfected cells and the persistence of expression. A: With pT2K-CAGGS-GFP plasmid concentration constant at 1.2 mg/mL, the transfected proportion was significantly increased when pT2K-CAGGS-GFP:pCAGGS-T2TP ratios were at 1:1/2 (*P <0.05) and 1:1/4 (**P <0.01) and decreased with 1:1/8 (#P <0.05) and 1:0 (###P <0.001) (all compared to1:1 ratio). B: GFP signal for co-electroporated plasmids (1:1 ratio) was retained in vivo at 7 days compared to 1 day after electroporation whereas,in the absence of transposase (1:0 ratio), the significantly lower expression level at 1 day (as in A) declined even further by 7 days (*P <0.05).

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yet the most rostral NC cells were inefficiently transfected. Corre-lating with this stage of inefficient transfection, transmissionelectron microscopy and live imaging show that some NC cellshave separated from the apical (luminal) surface and have losttheir apical cell-cell adhesive junctions typical of neural epithelialcells hours before exit from the neural tube (Newgreen and Gib-bins, 1982; Martins-Green and Erickson, 1987; Ahlstrom andErickson, 2009). Mesenchymal cells are notoriously difficult toelectroporate (Oberg et al., 2002; Scaal et al., 2004) because thecurrent path can circumnavigate cells rather than pass throughthem. It is likely that a NC cell with absence of epithelial-like api-

cal adhesions will evade transfection for the same reason. Wepropose that cells with this loss of apical adhesion continuityshould be defined as mesenchymal, therefore the stage of loss ofapical junctional integrity by a pre-migratory NC cell is the effec-tive stage of epithelium-mesenchyme transition (EMT) for thatcell even though this is well before overt emigration. We proposethat the term delamination, which is often used interchangeablywith EMT, be reserved to specify the stage of overt emigration,when an already mesenchymal NC cell leaves the confines of theneural tube.

Conclusions on Avian In Ovo ElectroporationParameters

For electroporation of mid-trunk level E2 neural tube withgenome-integrating plasmids at stated electrical parameters wesuggest:

1. It is unnecessary to use high plasmid concentrations,in this case higher than about 1.25 mg/mL.

2. No more than 3 pulses need to be used for maximumunilateral electroporation.

3. Bilateral electroporation should be used if the aim isto achieve the greatest total number of transfectedcells, although there is a lower efficiency per side.

4. Similar number of bilateral pulses (or slightly fewersecond pulses) should be used to produce similartransfection efficiencies on each side.

5. For higher local density of transfection and the pres-ence of an untransfected control side, unilateral elec-troporation is preferred.

6. For highest transfection with pT2K-CAGGS-GFP pluspCAGGS-T2TP genome-integrating plasmids, ratiosshould be optimised; we suggest a ratio of 4:1.

7. For efficient transfection of the earliest migratingtrunk NC cells, electroporation should occur morethan 8 hr prior to the time of nominal migration onsetat the specific level of interest (i.e., at least 6 somite-widths caudal to the position of onset of overt emigra-tion of NC cells at the time of electroporation).

Experimental Procedures

Embryo Preparation

Fertilised quail (Coturnix coturnix japonica) eggs were acquiredfrom Lago Game Supplies, Victoria, Australia. Eggs were storedat 14�C, and then incubated at 38�C and 60% humidity for peri-ods defined as embryonic (E) days. Embryos were staged bysomite counts and Hamburger and Hamilton stages (HH) (Ham-burger and Hamilton, 1951). Eggs were windowed to expose theembryo (Simkin et al., 2009).

Plasmids

The plasmid construct pT2K-CAGGS-GFP flanked by the recog-nition sequences for the transposon Tol2, and the Tol2 transpo-sase enzyme plasmid pCAGGS-T2TP, were obtained from ProfYoshiko Takahashi (Nara, Japan). The latter construct allows thefirst construct to be randomly and stably integrated into the host

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Fig. 3. NC cells electroporated shortly before onset of migration arenot efficiently transfected. NC cell outgrowth (red¼nuclei) and distribu-tion of GFP-expressing cells (green) from developmentally younger (A,C) and older (B, D) neural tubes (NT; see Fig. 2) are shown, with caudal(A, B) and rostral levels (C, D). E: Counts of transfected NC cells inzones I (proximal) to IV (distal). The proportion of transfected NC cellswas lower at the rostral end compared to the caudal end of young andold NCC outgrowths at zone III (zzzP <0.001 for young, nnn P <0.001 forold) and IV (DDDP <0.001 for young and ��� P <0.001 for old). Thisreduction was found progressively in the distal NC cells from the rostralend; young rostral NC (***P <0.001 zone II vs. I; III vs. I and II; IV vs. I,II, and III) and old rostral NT (yyP <0.01 zone II vs. I; yyy P <0.001 III vs.I and II; ### P <0.001 IV vs. I and II). In addition, at zone III, the pro-portion of transfected NC cells was also lower on old rostral comparedto young rostral ends (a P <0.05) suggesting the reduction was moreextreme in the older specimens.

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genome (Sato et al., 2007). Unless noted otherwise, the two plas-mids were mixed at equal concentrations and this was used atdilutions mentioned in the text. In a further series to investigatethe effect of plasmid ratio, the pT2K-CAGGS-GFP concentrationwas held constant at 1.2 mg/mL and the concentration of thepCAGGS-T2TP was varied as described.

Electroporation

Quail embryos at embryonic day (E) E1.8 to E2.5 (HH12þ to 15;or 17 to 26 somites) were exposed in ovo, and a glass micropip-ette was used to make a single injection of construct mix into thelumen of the neural tube caudal to the last somite. Immediatelyfollowing injection, gold electrodes were placed on the vitellinemembrane on either side of the embryo. Electrodes were L-shaped, 2 mm long and 0.5 mm diameter with separation of3 mm. These were aligned parallel with the embryonic axis (Fig.4, inset a) or, for a smaller electroporation field, with only the cir-cular electrode tips contacting the vitelline membrane (Fig. 4,inset b). An Electro Square PoratorTM (ECM 830, BTX, Gene-tronics, Inc.) was used to administer pulses to neural tube regions.For unilateral electroporation, conditions were 25 V, pulses of 50msec duration at 1-sec intervals with 1, 3, or 6 pulses as described(Simkin et al., 2009). In bilateral electroporations, the same elec-trical parameters were delivered, 3 pulse directed towards theright side, then by reversing the electrode polarity, 3 pulsestowards the left (termed 3þ3), or 1 pulse right, 5 pulses left(termed 1þ5), or 5 pulses right, 1 pulse left (termed 5þ1). Afterelectroporation, several drops of Tyrode’s solution were appliedto the embryo, the hole in the shell sealed with adhesive tape andthe egg returned to the humidified incubator for 1 day. To test

longer term retention, embryos were maintained until E9. Eachparameter test used neural tube cell counting data pooled from atleast three electroporation sessions.

Quantifying Electroporation Efficiency: Neural TubeIsolation, Dissociation, and Cell Counting

After 1 or 7 days further incubation, embryos (aged approx. E3and E9) were removed and placed in Ham’s F12 tissue culturemedium. Figure 4 shows schematically the quantification of elec-troporation efficiency. The GFP-positive region was identifiedand imaged with a Leica MZFLIII fluoresescent dissecting micro-scope and DC200 camera. A region of neural tube 5 somites inlength centred on the GFP maximum was dissected out with itssurrounding mesodermal tissues using sharpened tungsten nee-dles (Newgreen and Murphy, 2000). In cases of unilateral electro-poration, the entire neural tube was isolated from the aboveregion, and cells dissociated and counted (below). In cases ofbilateral electroporation, the neural tube was first bisected longi-tudinally whilst still in the embryo to ensure correct identificationof left and right halves, and each side processed separately.

These tissue segments were placed in Dispase II (2 mg/mL inF12, Roche, Castle Hill, NSW) for 20 min at 37�C, then each neu-ral tube was isolated from all surrounding tissues (Newgreen andMurphy, 2000) and placed into 100 mL of F12 in individualEppendorf tubes. Following this, 50 mL of 50 mM EGTA and 20mL of 0.05% trypsin (Roche) was added to each neural tube for 10min. The tissue was then triturated to dissociate the cells. Forcounts of electroporation efficiency, 12 mL of the dissociation cellsuspension was placed in separate Terasaki-type HLA plate wells(Nunc, Denmark). A fluorescent Olympus IX70 microscope

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Fig. 4. Neural tube electroporation and cell counting procedure. The GFP transfected region is indicated in green. The bottom inset shows awhole mount embryo with unilateral expression of GFP in the neural tube. The top inset shows a transverse section with unilateral expression ofGFP in the neural tube. Nuclei are labelled with DAPI (blue). NT, neural tube; Som, somite

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(Tokyo, Japan) was used to image GFP-positive and GFP-negative cells for cell counting using a Spot Monochrome cameramodel 2.1.1 with Image-Pro Plus 4.5 (MediaCybernetics, SilverSpring, MD) and Image-Pro-Analyser 6.1 (MediaCybernetics).

Neural Crest Cell Culture

Neural tubes of quail embryos of 14 to 17 somites age (HH11þ to12þ, about E1.8; termed “young”) and 20 to 22 somites age(HH13þ to 14, about E2, termed “old”) were bilaterally electropo-rated (3þ3) with 25 V, 50-msec duration pulses at 1-sec intervalsat the caudal-most somites and segmental plate levels (Fig. 5A,B). After up to 2 hr incubation in ovo, the electroporated neuraltube was dissected out with dispase as above such that the sameaxial level caudal from somite 19 (or somite 19’s projected level)was obtained at different developmental time points in relationto the onset of NC cell migration. These were explanted ontofibronectin-coated (Sigma, St. Louis, MO; 20 mg/mL) 3-cm dishes(Sarstedt, Melbourne) and NC cells allowed to migrate from theexplants for 1 day (Newgreen and Murphy, 2000). The cultureswere then fixed with 4% paraformaldehyde in PBS, labelled forNC marker Sox-E (rabbit anti-Sox-9, also recognising Sox-8 andSox-10, 1/2500; from Dr. Craig Smith, MCRI), GFP (goat anti-GFP, 1/400, Rockland), and DAPI (1 mg/mL, Sigma). Secondaryantibodies were donkey anti-rabbit: Alexa594 (1/2,000; Invitro-gen/Molecular Probes A21207) and donkey anti-goat: Alexa488(1/400; Invitrogen/Molecular Probes A11015). These explantswere evaluated microscopically. To count transfected NC cells,two rectangles were laid over images of each NC outgrowth, atabout one-somite width from the rostral and the caudal ends andperpendicular to the dorsal edge of the neural tube and extendingout to the most distant NC cell (Fig. 5C). These rectangles weremarked into four zones, I being most proximal to the neural tubeand IV most distal (Fig. 5D). The total number of cells and thenumber expressing GFP in each zone were recorded.

Statistics

Unless specified, data were expressed as mean6 standard error ofmean (SEM). All statistical tests were performed using GraphPadPrism version 6. A difference between two groups was determinedusing a two-tailed Student’s t-test. For differences among multi-ple groups, statistical comparisons were performed using one-way analyses of variance (one-way ANOVA) followed with Fish-er’s LSD post-test. A P value of <0.05 was considered significant.

AcknowledgmentsThis study was approved by the Royal Children’s Hospital AnimalEthics Committee, permits A596 and A650 and MCRI InstitutionalBiosafety Committee Certificate 127/2008. Dr. Craig Smith (MCRI)supplied the SoxE antibody and Prof. Yoshiko Takahashi suppliedthe Tol2 expression constructs. This work was performed withNHMRC grant 607379 and we acknowledge the Victorian Govern-ment’s Operational Infrastructure Support Program to MCRI.

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Fig. 5. Electroporation of NC cells and counting of GFP-labelled NC cells in vitro. Bilateral electroporation of young (A) and old (B) neural tubes(NT) in relation to somite (Som) levels. The open arrow indicates the caudal limit of overt NC cell migration at the time of electroporation. The linesindicate rostral (r) and caudal (c) limits of the neural tube explanted. C: Explant after 20 hr in vitro, showing NC cell (NCC) outgrowth with rostro-caudal (r,c) and dorso-ventral (d, v) axes indicated. Rectangles show the selected area for NC cell counting. D: Detail of NC cell outgrowth show-ing cell counting zones from I, closest to the neural tube, to IV, most distant.

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