Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC3105949&req=5

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.

Mentions: We next looked at the composition of the 111 responders in this group to determine the cellular activities affected. GO Tree Machine (GOTM) analysis was used to identify biological processes that were significantly enriched in this population. The biological function 'cellular component organisation' was significantly affected by exposure to current + GFP. Within this category 'regulation of cellular component organisation', 'organelle organisation' and 'cytoskeletal organisation' were significantly affected. These changes in 'organelle organisation' included the subterms 'chromatin organisation' and 'chromatin modification', which were significantly over-represented in the tissue exposed to current + GFP. This indicates that current + GFP may affect the cellular organisation (Figure 4A). This finding was also identified by Ingenuity Pathway Analysis (IPA). Using IPA, the most significant biological functions within the list of responders are 'cell-cell signalling and interaction', 'cellular assembly and organisation' and 'cellular function and maintenance' (Figure 4B). 'Cell death' is the ninth most significant pathway, with 9 out of the 111 genes found to be involved in this process. However, the changes in these apoptotic genes include ones that could both promote and reduce apoptosis (Figure 4B).


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

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

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

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

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.
Mentions: We next looked at the composition of the 111 responders in this group to determine the cellular activities affected. GO Tree Machine (GOTM) analysis was used to identify biological processes that were significantly enriched in this population. The biological function 'cellular component organisation' was significantly affected by exposure to current + GFP. Within this category 'regulation of cellular component organisation', 'organelle organisation' and 'cytoskeletal organisation' were significantly affected. These changes in 'organelle organisation' included the subterms 'chromatin organisation' and 'chromatin modification', which were significantly over-represented in the tissue exposed to current + GFP. This indicates that current + GFP may affect the cellular organisation (Figure 4A). This finding was also identified by Ingenuity Pathway Analysis (IPA). Using IPA, the most significant biological functions within the list of responders are 'cell-cell signalling and interaction', 'cellular assembly and organisation' and 'cellular function and maintenance' (Figure 4B). 'Cell death' is the ninth most significant pathway, with 9 out of the 111 genes found to be involved in this process. However, the changes in these apoptotic genes include ones that could both promote and reduce apoptosis (Figure 4B).

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