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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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Experimental strategy. (A) Dissection of the ventral lateral midbrain (VLM) region. (i) In situ sagital view of a HH st16 electroporated embryo expressing pCAβ-IRES-Dmrt5 construct (dorsal view in the inset). Red lines indicate the midbrain (Mb) region, which was dissected out. (ii-iv) Coronal sections of midbrain. White lines mark the VLM region. This region was isolated from (ii) control embryos (VLM), (iii) VLM exposed to current (VLMi), (iv) VLM exposed to current + GFP (VLMg), and VLM exposed to current + Dmrt5 (VLMd; image not shown) for investigation of transcriptional profiles by microarray analysis. (B) Tissue processing and microarray analysis. Six VLM tissues were pooled for each biological replicate, and three biological replicates were used for each condition. cDNA was isolated from these pools and hybridised to the Affymetrix Chicken Genome Array. Following MAS5, normalisation and filtering, genes whose expression differed significantly between the wild type (WT; VLM) and VLMi, VLMg and VLMd were identified by one-way ANOVA.
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Figure 1: Experimental strategy. (A) Dissection of the ventral lateral midbrain (VLM) region. (i) In situ sagital view of a HH st16 electroporated embryo expressing pCAβ-IRES-Dmrt5 construct (dorsal view in the inset). Red lines indicate the midbrain (Mb) region, which was dissected out. (ii-iv) Coronal sections of midbrain. White lines mark the VLM region. This region was isolated from (ii) control embryos (VLM), (iii) VLM exposed to current (VLMi), (iv) VLM exposed to current + GFP (VLMg), and VLM exposed to current + Dmrt5 (VLMd; image not shown) for investigation of transcriptional profiles by microarray analysis. (B) Tissue processing and microarray analysis. Six VLM tissues were pooled for each biological replicate, and three biological replicates were used for each condition. cDNA was isolated from these pools and hybridised to the Affymetrix Chicken Genome Array. Following MAS5, normalisation and filtering, genes whose expression differed significantly between the wild type (WT; VLM) and VLMi, VLMg and VLMd were identified by one-way ANOVA.

Mentions: To investigate the effects of the electric current and exogenous DNA expression associated with in ovo electroporation on endogenous gene expression of the developing chicken embryo, we used microarray analysis to compare the gene expression profile of four pooled samples of ventral lateral midbrain (VLM) tissue. The four samples were unelectroporated VLM (VLM), VLM exposed to electric current only (VLMi), VLM exposed to expression of GFP and electric current (VLMg) and VLM exposed to exogenous expression of a regulatory gene, Dmrt5 (Doublesex and Mab related transcription factor like 5) and electric current (VLMd) (Figure 1). The current was generated with an ECM830 Electro Square Porator (5 × 50-ms pulses of 12 V). The GFP construct used was pCAβ-IRES-GFP. Electroporation was carried out at HH st10, and embryos were collected 24 hours later. Thus, our experiment was specifically designed to interrogate the set of genes 'stably' regulated following electroporation, rather than those responding to the initial treatment. We reasoned that genes identified at this 24 hour time point would be more likely to have a significant contribution to altering long-term cell identity as opposed to those genes whose expression changed rapidly in the minutes following electrical insult.


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

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

Experimental strategy. (A) Dissection of the ventral lateral midbrain (VLM) region. (i) In situ sagital view of a HH st16 electroporated embryo expressing pCAβ-IRES-Dmrt5 construct (dorsal view in the inset). Red lines indicate the midbrain (Mb) region, which was dissected out. (ii-iv) Coronal sections of midbrain. White lines mark the VLM region. This region was isolated from (ii) control embryos (VLM), (iii) VLM exposed to current (VLMi), (iv) VLM exposed to current + GFP (VLMg), and VLM exposed to current + Dmrt5 (VLMd; image not shown) for investigation of transcriptional profiles by microarray analysis. (B) Tissue processing and microarray analysis. Six VLM tissues were pooled for each biological replicate, and three biological replicates were used for each condition. cDNA was isolated from these pools and hybridised to the Affymetrix Chicken Genome Array. Following MAS5, normalisation and filtering, genes whose expression differed significantly between the wild type (WT; VLM) and VLMi, VLMg and VLMd were identified by one-way ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Experimental strategy. (A) Dissection of the ventral lateral midbrain (VLM) region. (i) In situ sagital view of a HH st16 electroporated embryo expressing pCAβ-IRES-Dmrt5 construct (dorsal view in the inset). Red lines indicate the midbrain (Mb) region, which was dissected out. (ii-iv) Coronal sections of midbrain. White lines mark the VLM region. This region was isolated from (ii) control embryos (VLM), (iii) VLM exposed to current (VLMi), (iv) VLM exposed to current + GFP (VLMg), and VLM exposed to current + Dmrt5 (VLMd; image not shown) for investigation of transcriptional profiles by microarray analysis. (B) Tissue processing and microarray analysis. Six VLM tissues were pooled for each biological replicate, and three biological replicates were used for each condition. cDNA was isolated from these pools and hybridised to the Affymetrix Chicken Genome Array. Following MAS5, normalisation and filtering, genes whose expression differed significantly between the wild type (WT; VLM) and VLMi, VLMg and VLMd were identified by one-way ANOVA.
Mentions: To investigate the effects of the electric current and exogenous DNA expression associated with in ovo electroporation on endogenous gene expression of the developing chicken embryo, we used microarray analysis to compare the gene expression profile of four pooled samples of ventral lateral midbrain (VLM) tissue. The four samples were unelectroporated VLM (VLM), VLM exposed to electric current only (VLMi), VLM exposed to expression of GFP and electric current (VLMg) and VLM exposed to exogenous expression of a regulatory gene, Dmrt5 (Doublesex and Mab related transcription factor like 5) and electric current (VLMd) (Figure 1). The current was generated with an ECM830 Electro Square Porator (5 × 50-ms pulses of 12 V). The GFP construct used was pCAβ-IRES-GFP. Electroporation was carried out at HH st10, and embryos were collected 24 hours later. Thus, our experiment was specifically designed to interrogate the set of genes 'stably' regulated following electroporation, rather than those responding to the initial treatment. We reasoned that genes identified at this 24 hour time point would be more likely to have a significant contribution to altering long-term cell identity as opposed to those genes whose expression changed rapidly in the minutes following electrical insult.

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