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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

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.
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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.

Mentions: Given the high number of patterning marker genes that are well described for the VLM, we were able to analyse the effects of current and expression of GFP on patterning of this region. The VLM is lateral to the floor plate, in the Nkx6.2 expressing region; therefore, we expected markers such as Nkx6.2 to be present in the microarray. Analysis of the transcriptional profile obtained for the VLM was reflective of the known genetic profile of the region and the expression levels of the ten marker genes were similar in the VLM, VLMi and VLMg samples (Figure 6A).


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

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

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

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

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.
Mentions: Given the high number of patterning marker genes that are well described for the VLM, we were able to analyse the effects of current and expression of GFP on patterning of this region. The VLM is lateral to the floor plate, in the Nkx6.2 expressing region; therefore, we expected markers such as Nkx6.2 to be present in the microarray. Analysis of the transcriptional profile obtained for the VLM was reflective of the known genetic profile of the region and the expression levels of the ten marker genes were similar in the VLM, VLMi and VLMg samples (Figure 6A).

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