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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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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.
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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.

Mentions: The distribution of gene expression values across the whole microarray was monitored before and after normalisation by quantile grouping (box plots) and was found to be similar for each set of biological replicates (data not shown). The variation in biological replicates was also analysed by principal component analysis (PCA), which measures the variation in expression levels between the microarrays. PCA showed all biological replicates cluster together, demonstrating that the biological replicate datasets are similar and reproducible. As well as biological replicates clustering together, three experimental conditions, VLM, VLMi and VLMg, cluster together, indicating there is little difference in gene expression between these three conditions. In contrast, exogenous expression of the regulatory gene Dmrt5 caused the VLMd samples to cluster separately from the other conditions (Figure 2). This preliminary analysis of global gene expression levels indicates that exposure to current or current + GFP has little effect on endogenous gene expression, whilst addition of a regulatory gene causes a much larger change in endogenous gene expression.


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

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

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

License
Show All Figures
getmorefigures.php?uid=PMC3105949&req=5

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.
Mentions: The distribution of gene expression values across the whole microarray was monitored before and after normalisation by quantile grouping (box plots) and was found to be similar for each set of biological replicates (data not shown). The variation in biological replicates was also analysed by principal component analysis (PCA), which measures the variation in expression levels between the microarrays. PCA showed all biological replicates cluster together, demonstrating that the biological replicate datasets are similar and reproducible. As well as biological replicates clustering together, three experimental conditions, VLM, VLMi and VLMg, cluster together, indicating there is little difference in gene expression between these three conditions. In contrast, exogenous expression of the regulatory gene Dmrt5 caused the VLMd samples to cluster separately from the other conditions (Figure 2). This preliminary analysis of global gene expression levels indicates that exposure to current or current + GFP has little effect on endogenous gene expression, whilst addition of a regulatory gene causes a much larger change in endogenous gene expression.

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