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. 2011 Apr 28:6:17.
doi: 10.1186/1749-8104-6-17.

Effects of in ovo electroporation on endogenous gene expression: genome-wide analysis

Emma K Farley  1 , Emily Gale, David Chambers, Meng Li
Affiliations

Effects of in ovo electroporation on endogenous gene expression: genome-wide analysis

Emma K Farley et al. Neural Dev. .

Abstract

Background: In ovo electroporation is a widely used technique to study gene function in developmental biology. Despite the widespread acceptance of this technique, no genome-wide analysis of the effects of in ovo electroporation, principally the current applied across the tissue and exogenous vector DNA introduced, on endogenous gene expression has been undertaken. Here, the effects of electric current and expression of a GFP-containing construct, via electroporation into the midbrain of Hamburger-Hamilton stage 10 chicken embryos, are analysed by microarray.

Results: Both current alone and in combination with exogenous DNA expression have a small but reproducible effect on endogenous gene expression, changing the expression of the genes represented on the array by less than 0.1% (current) and less than 0.5% (current + DNA), respectively. The subset of genes regulated by electric current and exogenous DNA span a disparate set of cellular functions. However, no genes involved in the regional identity were affected. In sharp contrast to this, electroporation of a known transcription factor, Dmrt5, caused a much greater change in gene expression.

Conclusions: These findings represent the first systematic genome-wide analysis of the effects of in ovo electroporation on gene expression during embryonic development. The analysis reveals that this process has minimal impact on the genetic basis of cell fate specification. Thus, the study demonstrates the validity of the in ovo electroporation technique to study gene function and expression during development. Furthermore, the data presented here can be used as a resource to refine the set of transcriptional responders in future in ovo electroporation studies of specific gene function.

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Figures

Figure 1
Figure 1
Experimental strategy. (A) Dissection of the ventral lateral midbrain (VLM) region. (i) In situ sagital view of a HH st16 electroporated embryo expressing pCAβ-IRES-Dmrt5 construct (dorsal view in the inset). Red lines indicate the midbrain (Mb) region, which was dissected out. (ii-iv) Coronal sections of midbrain. White lines mark the VLM region. This region was isolated from (ii) control embryos (VLM), (iii) VLM exposed to current (VLMi), (iv) VLM exposed to current + GFP (VLMg), and VLM exposed to current + Dmrt5 (VLMd; image not shown) for investigation of transcriptional profiles by microarray analysis. (B) Tissue processing and microarray analysis. Six VLM tissues were pooled for each biological replicate, and three biological replicates were used for each condition. cDNA was isolated from these pools and hybridised to the Affymetrix Chicken Genome Array. Following MAS5, normalisation and filtering, genes whose expression differed significantly between the wild type (WT; VLM) and VLMi, VLMg and VLMd were identified by one-way ANOVA.
Figure 2
Figure 2
Principal component analysis to identify genome-wide transcriptional variation caused by in ovo electroporation. This plot shows the variation between the samples; each dot represents the global gene expression of a single microarray. The greatest variation is measured on the x-axis, then the y- and z-axes, respectively. Wild-type VLM, VLMi and VLMg samples cluster together on the x-axis, indicating that there is little variation in the gene expression between these three conditions. VLMd, in which the regulatory gene Dmrt5 is exogenously expressed, clusters separately, indicating larger genome-wide variation between this sample and the others.
Figure 3
Figure 3
Genes showing differential expression in response to current. Genes showing differential expression when exposed to current and their fold change.
Figure 4
Figure 4
Effect of electric current + GFP expression on endogenous gene expression of the VLM. (A) Genes showing differential expression when exposed to current + GFP and their fold changes. For gene names refer to Table 3. (B) GOTM analysis showing biological function and expected and observed number of genes in each category compared to the number expected from a random set of 111 genes. (C) IPA functional analysis showing the top ten biological functions enriched in the VLMg compared to VLM samples.
Figure 5
Figure 5
Ingenuity Pathway Analysis toxicity analysis. The genes showing differential expression upon exposure to current + GFP are not significantly involved in any type of toxicity. The bar chart shows the percentage of genes involved in each type of toxicity (left axis). Numbers above the bars are the number of genes that would equate to 100% for each type of toxicity. The orange points show the -log(P-value) (right axis) for each category, and thus indicate whether a significant number of genes are found within a category to suggest an actual toxicity affect. A -log(P-value) of 1.3 would be considered significant; all -log(P-values) are lower than 1.3 and therefore not significant.
Figure 6
Figure 6
Regional identity of VLM unaffected by current alone or current + GFP. (A) Expression profile of ten marker genes of the VLM obtained from the microarray analysis of VLM (wild type (WT)), VLMi and VLMg. In all conditions, marker genes are seen at the expected levels, and there is little difference between the three conditions. (B) Box plot showing the normalised expression values for all ten marker genes. All conditions have the same median and error bars overlap, indicating that the ten marker genes are not significantly differentially expressed between the three conditions. This shows the regional identity is not significantly affected by exposure to current or current + GFP. Statistical analysis using one-way ANOVA also shows that these marker genes are not differentially expressed.
Figure 7
Figure 7
Venn diagram showing overlap between genes differentially expressed in all three conditions. Venn diagram showing annotated endogenous genes that are differentially expressed in all three conditions - current (VLMi), current + GFP (VLMg) and current + Dmrt5 (VLMd) - and the fold change range for each condition.
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