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

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

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

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.

Mentions: Three genes are affected in all experimental conditions, current, current + GFP and current + Dmrt5 (Figure 7). These are Hnrnpk (Heterogeneous nuclear ribonucleoprotein K), Chd1 (Chromodomain-helicase-DNA-binding protein 1) and Txnl1 (Thioredoxin-like protein 1). Hnrnpk is an RNA binding protein known to associate with pre-mRNAs in the nucleus and influences RNA processing, metabolism and transport. It is involved in maintaining cellular ATP levels in stress conditions, possibly by protecting its target mRNAs [38]. Chd1 is a chromatin remodelling enzyme that regulates transcription [39], and Txnl1 is a redox sensor [40].


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

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

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

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

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.
Mentions: Three genes are affected in all experimental conditions, current, current + GFP and current + Dmrt5 (Figure 7). These are Hnrnpk (Heterogeneous nuclear ribonucleoprotein K), Chd1 (Chromodomain-helicase-DNA-binding protein 1) and Txnl1 (Thioredoxin-like protein 1). Hnrnpk is an RNA binding protein known to associate with pre-mRNAs in the nucleus and influences RNA processing, metabolism and transport. It is involved in maintaining cellular ATP levels in stress conditions, possibly by protecting its target mRNAs [38]. Chd1 is a chromatin remodelling enzyme that regulates transcription [39], and Txnl1 is a redox sensor [40].

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