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

Farley EK, Gale E, Chambers D, Li M - Neural Dev (2011)

Bottom Line: 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.However, no genes involved in the regional identity were affected.The analysis reveals that this process has minimal impact on the genetic basis of cell fate specification.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Clinical Sciences Centre, Imperial College London, W12 0NN, UK. e.farley07@csc.mrc.ac.uk

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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Related in: MedlinePlus

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.
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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.

Mentions: To identify if any of the changes in gene expression upon exposure to current + GFP elicit a toxic affect, such as oxidative stress or apoptosis, IPA toxicity analysis was used (Figure 5). This analysis found some genes involved in pro-apoptosis and p53 signalling in the set of 111 responders. However, the number found is less than would be expected to occur in a random set of 111 genes, suggesting this is not statistically significant. Thus, in ovo electroporation does not induce toxic effects within the embryo as defined by the pathway analysis software.


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

Farley EK, Gale E, Chambers D, Li M - Neural Dev (2011)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105949&req=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.
Mentions: To identify if any of the changes in gene expression upon exposure to current + GFP elicit a toxic affect, such as oxidative stress or apoptosis, IPA toxicity analysis was used (Figure 5). This analysis found some genes involved in pro-apoptosis and p53 signalling in the set of 111 responders. However, the number found is less than would be expected to occur in a random set of 111 genes, suggesting this is not statistically significant. Thus, in ovo electroporation does not induce toxic effects within the embryo as defined by the pathway analysis software.

Bottom Line: 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.However, no genes involved in the regional identity were affected.The analysis reveals that this process has minimal impact on the genetic basis of cell fate specification.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Clinical Sciences Centre, Imperial College London, W12 0NN, UK. e.farley07@csc.mrc.ac.uk

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.

Show MeSH
Related in: MedlinePlus