Modified Whole-Mount In situ Hybridization Protocol for the Detection of Transgene Expression in Electroporated Chick Embryos

Background In vivo electroporation has been extensively used as an effective means of DNA transfer for analyzing gene function as well as gene regulation in developmental systems. In any of these two types of studies, the correct spatial and temporal expression of the electroporated transgene can only be accurately assessed by in situ hybridization. Methodology/Principal Findings While analyzing transgene expression in electroporated chicken embryos, we verified that transgene riboprobes cross-hybridized with the exogenous plasmid DNA when embryos were processed by conventional whole-mount in situ hybridization (WISH). Conclusions/Significance Here we describe a modification to the WISH protocol that is essential to prevent DNA cross-hybridization and to specifically detect transgene mRNA transcripts in electroporated embryos. Our optimized WISH procedure can be applied not only to electroporated chick embryos but also to other embryos or adult tissues that have been transfected with large amounts of reporter- or expression construct DNA.


Introduction
In vivo electroporation is a very effective technique for introducing DNA into cells of various animal models, including Drosophila [1], ascidians [2], zebrafish [3], axolotl [4], Xenopus [5], chick (reviewed in [6]) and mouse [7]. This method makes use of electric pulses to create transient pores in the plasma membrane through which the negatively charged DNA molecules enter the cell. The electroporation of living embryos has been extensively used in gain-of-function studies, for which the expression vectors carry a ubiquitous promoter driving the transgene transcription, as well as in loss-of-function approaches, such as those using siRNA or morpholinos (reviewed in [6]). In addition to gene function, the electroporation method has also emerged as a powerful tool to analyze gene regulatory sequences, particularly in chicken (reviewed in [8,9]) and ascidians [2] embryos.
In most functional studies, the expression construct is coelectroporated with a fluorescent reporter plasmid to evaluate the efficiency of transfection in live tissues or embryos. Since both vectors carry the same regulatory sequences, reporter fluorescence is also used to indirectly monitor the expression of the exogenous gene. However, the correct transcription of the transgene can only be accurately assessed by in situ hybridization. Fluorescent reporters are also broadly used as readout of enhancer activity in studies of gene regulation. However, fluorescence becomes visible at least two to three hours after induction of transcription. Therefore, to determine exactly when and where a certain enhancer is active, reporter mRNA localization must be investigated. Here we describe a modification of the conventional whole-mount in situ hybridization (WISH) procedure that proved to be crucial for the correct detection of transgene transcripts in electroporated embryos.

Results
In order to investigate the transcription pattern of an overexpressed transgene, chick embryos were electroporated with the pCAGGS-RFP construct, which carries the cDNA of monomeric red fluorescent protein (RFP; [10]) under the control of the CAGGS ubiquitous promoter [11], and the pattern of fluorescence was compared with the localization of RFP transcripts. When embryos were processed by conventional whole-mount in situ hybridization (WISH; [12,13]), the RFP antisense riboprobe was detected in the red fluorescent cells ( Figure 1A,B). However, a similar pattern was seen not only in embryos treated with RNase A before hybridization ( Figure 1C,D) but also in those hybridized with the RFP sense probe ( Figure 1E,F). The digestion of RNA transcripts with RNase A as well as the hybridization of the sense probe are expected to work as controls for background staining. These observations suggested that the RFP probes were hybridizing with the electroporated DNA of the reporter construct. This DNA cross-hybridization was transgene-specific and was not observed when transgene-unrelated probes were used, such as those against eGFP (data not shown) and left-side specific genes [14].
To avoid cross-hybridization, we tested different enzymatic digestions and stringency conditions. We could eliminate the detection of electroporated DNA in three situations: (i) DNase I digestion before hybridization, to degrade the electroporated DNA, (ii) RNase H treatment after hybridization, to eliminate the DNA-RNA hybrids (Figure S1A-D), and (iii) probe hybridization at 55uC, to avoid DNA denaturation ( Figure S1E-H). Among them, the first condition proved less disturbing to the embryo integrity and to cause less background. After DNase treatment, the RFP antisense probe still labeled the RFP-expressing cell population ( Figure 1G,H), but not in embryos digested also with RNase A ( Figure 1I,J), whereas the RFP sense probe was no longer detected ( Figure 1K,L). These results indicate that DNase digestion is essential to avoid DNA cross-hybridization and to exclusively detect transgene transcripts in embryos electroporated with expression constructs.
The modification of the WISH protocol proved to be indispensable in gene regulation studies using tissue-specific reporters. During our study of the transcriptional regulation of chick Cerberus (cCer) in early development, cCer 59 genomic fragments were subcloned upstream the enhanced green fluorescent protein (eGFP) and cCer-eGFP constructs were introduced into chick embryos by electroporation [14]. The ubiquitous reporter pCAGGS-RFP was co-electroporated to label the populations of targeted cells. In embryos electroporated with the Cer0.4-eGFP reporter, which carries the complete regulatory region of the cCer gene (i.e., the 400 base pairs sequence upstream the ATG; [14]), eGFP fluorescence was restricted to the anterior mesendoderm ( Figure 2). However, when these embryos were processed by standard procedures for WISH, the eGFP antisense probe labeled not only the eGFP-expressing cells but also the RFPpositive cells (Figure 2A-D). The electroporated cells were also labeled by the eGFP sense probe ( Figure 2E-H), indicating once again that both probes were cross-hybridizing with the DNA of the eGFP reporter construct. The detection of plasmid DNA was eliminated in embryos treated with DNase I before probe hybridization: the eGFP antisense probe specifically labeled the eGFP fluorescent cells ( Figure 2I-L), whereas the eGFP sense probe was no longer detected ( Figure 2M-P). These observations demonstrate that the addition of a DNase digestion step to the WISH protocol is fundamental for the correct localization of tissue-specific reporter transcripts in enhancer studies. Our modified WISH procedure was particularly important for the expression analysis of silent reporter constructs, such as Cer0.12-eGFP, which carries the minimal promoter of the cCer gene (i.e., the 120 base pairs sequence upstream the ATG; [14]). In embryos co-electroporated with Cer0.12-eGFP and pCAGGS-RFP, eGFP fluorescence was undetectable ( Figure 3). However, the eGFP antisense probe was detected in the RFP-expressing cell population when embryos were processed by conventional WISH (Figure 3A-D). The absence of eGFP expression was revealed only in embryos treated with DNase I (Figure 3E-H).
In summary, our observations suggest that the mRNA expression of electroporated transgenes can only be accurately assessed if a DNase step is added to the standard WISH protocol. This modified procedure is especially crucial in reporter-our expression assays using tissue-specific enhancers.

Discussion
We have shown that, when electroporated embryos are processed by conventional WISH techniques for the detection of transgene expression, transgene riboprobes hybridize not only with the transgene mRNA transcripts but also with the plasmid DNA. One of the reasons for this cross-hybridization is the fact that the electroporated DNA is delivered in very large amounts (i.e., 100-200 nanograms per embryo) and can remain in cell nuclei for many days [15]. In contrast, in transgenic zebrafish, Xenopus or mouse embryos, transgene copies are much fewer and undetectable by standard WISH protocols (e.g. [14]). DNA crosshybridization may also be triggered by the stringency conditions generally used for WISH (i.e., hybridization at 70uC in 50% formamide), which can promote the denaturation of the electroporated plasmid DNA [16] and its hybridization with complementary riboprobes [17,18]. Indeed, we observed that reducing the hybridization temperature to 55uC was enough to avoid DNA cross-hybridization and confer specificity to the detection of eGFP transcripts in electroporated embryos (see Figure  S1E-H). The detection of transgene transcripts by WISH in electroporated embryos has been reported in studies using ubiquitous expression vectors (e.g. [3,8]). In these type of experiments, since the transgene is transcribed in all targeted cells, its expression pattern coincides with the distribution of the plasmid DNA and, therefore, the hybridization of transgene probes with the electroporated DNA is imperceptible (see Figure 1). DNA crosshybridization becomes most evident in electroporation studies using tissue-specific enhancers (Figures 2 and 3). In any of these two types of assays, we have demonstrated that the binding of transgene probes to the electroporated DNA can be avoided when the embryos are treated with DNase I before hybridization.
In conclusion, we describe an optimized protocol for WISH that is crucial for the accurate detection of transgene expression in electroporated embryos. In addition to chick embryos, this modified procedure is applicable to other embryos and to adult tissues, such as those subject to gene therapy by electroporation [19]. Moreover, our WISH procedure may provide a reliable way to localize transgene expression whenever large amounts of naked plasmid DNA are transferred into tissues, not only by electroporation but also by other gene delivery methods.

Whole-mount in situ hybridization
Embryos were fixed overnight in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) plus 0.1% TweenH 20 (PBT) at 4uC, dehydrated though a series of methanol/PBT solutions (25%, 50%, 75% and 100% methanol), and stored at 220uC until hybridization. Fixed embryos were rehydrated and rinsed twice in PBT. At this point, embryos were either digested with DNase and/ or RNase, or kept in PBT. For DNA digestion, embryos were incubated with RNase-free DNase I (50 U/ml in DNase I buffer; Ambion) for 1h at 37uC. For the elimination of RNA transcripts, embryos were treated with DNase-free RNase A (100 mg/ml in PBT) for 1h at 37uC. The RNase A enzyme was inactivated with 0.5x standard saline citrate (SSC)/0.1%SDS for 10 min at room temperature.
All embryos were bleached in 6% hydrogen peroxide in PBT for 1h. Embryos were then rinsed 3 times in PBT for 5 min, digested with proteinase K (10 mg/ml in PBT) for 5 min at room temperature, washed once in 2 mg/ml glycine in PBT and twice in PBT for 5 min each, and post-fixed in 4% PFA/0.2% glutaraldehyde in PBT for 20 min at room temperature. Embryos were subsequently rinsed twice in PBT for 5 min and prehybridized at 70uC in hybridization solution (50% formamide, 5x SSC, pH 5, 0.1% TweenH 20, 50 mg/ml heparin, 50 mg/ml Torula RNA, 50 mg/ml salmon sperm DNA) for 2h. Embryos were then incubated overnight at 70uC in hybridization solution containing 500 ng/ml of denatured riboprobe. Riboprobes were generated by in vitro transcription in the presence of Digoxigenin-UTP (Roche Diagnostics). The antisense and sense probes span their entire coding sequences of eGFP and RFP and were synthesized from linearized pCS2-eGFP and pCAGGS-RFP plasmids, respectively. On the second day, embryos were washed twice in 50% formamide/4x SSC, pH 5/1% SDS and twice in 50% formamide/2x SSC, pH 5 for 30 min each. These post-hybridization washes were carried out at 55uC. Embryos were then rinsed three times for 5 min in MABT (100 mM maleic acid, 150 mM NaCl, pH 7.5, 0.1% TweenH), blocked for 2 h at room temperature in 10 % goat serum in MABT, and incubated overnight at 4uC in 1% goat serum in MABT with 1:5000 alkaline phosphatase-coupled anti-Digoxigenin antibody (Roche Diagnostics). On the third day, embryos were washed in MABT twice for 5 min and five more times for 1 h each. Embryos were then rinsed twice in NTMT (100 mM NaCl, 100 mM TrisHCl, pH 9.5, 50 mM MgCl2, 0.1% TweenH) for 15 min each, followed by the staining reaction in BM Purple (Roche Diagnostics) in the dark for 30 min to 12 h. Stained embryos were fixed overnight in 4% PFA in PBT, stored in PBT and photographed under a Leica MZ16FA stereomicroscope.