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3T3 cell lines stably expressing Pax6 or Pax6(5a)--a new tool used for identification of common and isoform specific target genes.

Kiselev Y, Eriksen TE, Forsdahl S, Nguyen LH, Mikkola I - PLoS ONE (2012)

Bottom Line: A majority of these were associated with the extracellular region.Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP.Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities.

View Article: PubMed Central - PubMed

Affiliation: Research Group of Pharmacology, Department of Pharmacy, University of Tromsø, Tromsø, Norway.

ABSTRACT
Pax6 and Pax6(5a) are two isoforms of the evolutionary conserved Pax6 gene often co-expressed in specific stochiometric relationship in the brain and the eye during development. The Pax6(5a) protein differs from Pax6 by having a 14 amino acid insert in the paired domain, causing the two proteins to have different DNA binding specificities. Difference in functions during development is proven by the fact that mutations in the 14 amino acid insertion for Pax6(5a) give a slightly different eye phenotype than the one described for Pax6. Whereas quite many Pax6 target genes have been published during the last years, few Pax6(5a) specific target genes have been reported on. However, target genes identified by Pax6 knockout studies can probably be Pax6(5a) targets as well, since this isoform also will be affected by the knockout. In order to identify new Pax6 target genes, and to try to distinguish between genes regulated by Pax6 and Pax6(5a), we generated FlpIn-3T3 cell lines stably expressing Pax6 or Pax6(5a). RNA was harvested from these cell lines and used in gene expression microarrays where we identified a number of genes differentially regulated by Pax6 and Pax6(5a). A majority of these were associated with the extracellular region. By qPCR we verified that Ncam1, Ngef, Sphk1, Dkk3 and Crtap are Pax6(5a) specific target genes, while Tgfbi, Vegfa, EphB2, Klk8 and Edn1 were confirmed as Pax6 specific target genes. Nbl1, Ngfb and seven genes encoding different glycosyl transferases appeared to be regulated by both. Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP. Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities.

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Real time quantitative PCR verifies differentially regulated genes in both the FlpIn-3T3 Pax6 and Pax6(5a) cell lines.Primers were designed for genes representative for the gene ontology terms “Extracellular matrix” (Adamts2, Tgfbi, Vegfa and Crtap), “Cell adhesion” (Ctgf, Cdh5, Ncam1 and Ngef), “Pathways in cancer” (Ephb2, Pdgfra, Pdgfrb, Ppp3ca, Prkcn and Vegfa) and “Regulation of transmission of nerve impulses” (Bdnf, Edn1, Klk8 and Ngfb) and used in qPCR. Results are means of three independent experiments and are presented as fold change in cDNA concentrations in the FlpIn-3T3 Pax6 and Pax6(5a) cell lines compared to the FlpIn-3T3 control cell line, after normalization against two housekeeping genes. The fold change values obtained for the same genes in the Illumina gene expression microarray are given in the table to the left of each graph for comparison.
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pone-0031915-g004: Real time quantitative PCR verifies differentially regulated genes in both the FlpIn-3T3 Pax6 and Pax6(5a) cell lines.Primers were designed for genes representative for the gene ontology terms “Extracellular matrix” (Adamts2, Tgfbi, Vegfa and Crtap), “Cell adhesion” (Ctgf, Cdh5, Ncam1 and Ngef), “Pathways in cancer” (Ephb2, Pdgfra, Pdgfrb, Ppp3ca, Prkcn and Vegfa) and “Regulation of transmission of nerve impulses” (Bdnf, Edn1, Klk8 and Ngfb) and used in qPCR. Results are means of three independent experiments and are presented as fold change in cDNA concentrations in the FlpIn-3T3 Pax6 and Pax6(5a) cell lines compared to the FlpIn-3T3 control cell line, after normalization against two housekeeping genes. The fold change values obtained for the same genes in the Illumina gene expression microarray are given in the table to the left of each graph for comparison.

Mentions: Quantitative real time PCR primers were designed for some of the genes associated with extracellular matrix (Tgfbi, Vegfa, Adamts-2 and Crtap) and cell adhesion (Ctgf, Cdh5 and Ncam). Some genes representing the KEGG pathway “Pathways in cancer” (Pdgfra, Pdgfrb, Vegfa, PPP3ca, EphB2 and Prkcn) and the gene ontology term “regulation of transmission of nerve impulses” (Bdnf, Ngfb, Klk8 and Edn1) were also included (Fig. 4). As well, we performed qPCR on some genes listed as Pax6(5a) specific target genes (Ncam1, Pdgfb, Ngef, Dkk3, Hist1h1c, Sphk1and Nbl1). When a cut-off of fold change (FC) 1.7 was used, most of the genes showed up- or downregulation as expected from the microarray result. However, Adamts2, Pdgfra, Ppp3ca and Bdnf did not seem to be regulated at all, and Prkcn value was borderline. According to microarray results Tgfbi, Vegfa, EphB2, Klk8 were expected to be regulated by Pax6 only, and this was confirmed by qPCR. Likewise, Crtap was downregulated and Ctgf was upregulated as expected by both Pax6 and Pax6(5a). We noticed that Crtap is much stronger downregulated by Pax6(5a) (74 fold) than by Pax6 (4 fold). Further, for several of the Pax6 specific target genes tested, real time PCR showed regulation by the Pax6(5a) isoform as well (e.g Edn1, Ngfb, Cdh5), even though the Edn1 gene in particular seemed to be much stronger regulated by Pax6 than Pax6(5a) (17 fold compared to 3.7 fold) (Fig. 4).


3T3 cell lines stably expressing Pax6 or Pax6(5a)--a new tool used for identification of common and isoform specific target genes.

Kiselev Y, Eriksen TE, Forsdahl S, Nguyen LH, Mikkola I - PLoS ONE (2012)

Real time quantitative PCR verifies differentially regulated genes in both the FlpIn-3T3 Pax6 and Pax6(5a) cell lines.Primers were designed for genes representative for the gene ontology terms “Extracellular matrix” (Adamts2, Tgfbi, Vegfa and Crtap), “Cell adhesion” (Ctgf, Cdh5, Ncam1 and Ngef), “Pathways in cancer” (Ephb2, Pdgfra, Pdgfrb, Ppp3ca, Prkcn and Vegfa) and “Regulation of transmission of nerve impulses” (Bdnf, Edn1, Klk8 and Ngfb) and used in qPCR. Results are means of three independent experiments and are presented as fold change in cDNA concentrations in the FlpIn-3T3 Pax6 and Pax6(5a) cell lines compared to the FlpIn-3T3 control cell line, after normalization against two housekeeping genes. The fold change values obtained for the same genes in the Illumina gene expression microarray are given in the table to the left of each graph for comparison.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285655&req=5

pone-0031915-g004: Real time quantitative PCR verifies differentially regulated genes in both the FlpIn-3T3 Pax6 and Pax6(5a) cell lines.Primers were designed for genes representative for the gene ontology terms “Extracellular matrix” (Adamts2, Tgfbi, Vegfa and Crtap), “Cell adhesion” (Ctgf, Cdh5, Ncam1 and Ngef), “Pathways in cancer” (Ephb2, Pdgfra, Pdgfrb, Ppp3ca, Prkcn and Vegfa) and “Regulation of transmission of nerve impulses” (Bdnf, Edn1, Klk8 and Ngfb) and used in qPCR. Results are means of three independent experiments and are presented as fold change in cDNA concentrations in the FlpIn-3T3 Pax6 and Pax6(5a) cell lines compared to the FlpIn-3T3 control cell line, after normalization against two housekeeping genes. The fold change values obtained for the same genes in the Illumina gene expression microarray are given in the table to the left of each graph for comparison.
Mentions: Quantitative real time PCR primers were designed for some of the genes associated with extracellular matrix (Tgfbi, Vegfa, Adamts-2 and Crtap) and cell adhesion (Ctgf, Cdh5 and Ncam). Some genes representing the KEGG pathway “Pathways in cancer” (Pdgfra, Pdgfrb, Vegfa, PPP3ca, EphB2 and Prkcn) and the gene ontology term “regulation of transmission of nerve impulses” (Bdnf, Ngfb, Klk8 and Edn1) were also included (Fig. 4). As well, we performed qPCR on some genes listed as Pax6(5a) specific target genes (Ncam1, Pdgfb, Ngef, Dkk3, Hist1h1c, Sphk1and Nbl1). When a cut-off of fold change (FC) 1.7 was used, most of the genes showed up- or downregulation as expected from the microarray result. However, Adamts2, Pdgfra, Ppp3ca and Bdnf did not seem to be regulated at all, and Prkcn value was borderline. According to microarray results Tgfbi, Vegfa, EphB2, Klk8 were expected to be regulated by Pax6 only, and this was confirmed by qPCR. Likewise, Crtap was downregulated and Ctgf was upregulated as expected by both Pax6 and Pax6(5a). We noticed that Crtap is much stronger downregulated by Pax6(5a) (74 fold) than by Pax6 (4 fold). Further, for several of the Pax6 specific target genes tested, real time PCR showed regulation by the Pax6(5a) isoform as well (e.g Edn1, Ngfb, Cdh5), even though the Edn1 gene in particular seemed to be much stronger regulated by Pax6 than Pax6(5a) (17 fold compared to 3.7 fold) (Fig. 4).

Bottom Line: A majority of these were associated with the extracellular region.Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP.Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities.

View Article: PubMed Central - PubMed

Affiliation: Research Group of Pharmacology, Department of Pharmacy, University of Tromsø, Tromsø, Norway.

ABSTRACT
Pax6 and Pax6(5a) are two isoforms of the evolutionary conserved Pax6 gene often co-expressed in specific stochiometric relationship in the brain and the eye during development. The Pax6(5a) protein differs from Pax6 by having a 14 amino acid insert in the paired domain, causing the two proteins to have different DNA binding specificities. Difference in functions during development is proven by the fact that mutations in the 14 amino acid insertion for Pax6(5a) give a slightly different eye phenotype than the one described for Pax6. Whereas quite many Pax6 target genes have been published during the last years, few Pax6(5a) specific target genes have been reported on. However, target genes identified by Pax6 knockout studies can probably be Pax6(5a) targets as well, since this isoform also will be affected by the knockout. In order to identify new Pax6 target genes, and to try to distinguish between genes regulated by Pax6 and Pax6(5a), we generated FlpIn-3T3 cell lines stably expressing Pax6 or Pax6(5a). RNA was harvested from these cell lines and used in gene expression microarrays where we identified a number of genes differentially regulated by Pax6 and Pax6(5a). A majority of these were associated with the extracellular region. By qPCR we verified that Ncam1, Ngef, Sphk1, Dkk3 and Crtap are Pax6(5a) specific target genes, while Tgfbi, Vegfa, EphB2, Klk8 and Edn1 were confirmed as Pax6 specific target genes. Nbl1, Ngfb and seven genes encoding different glycosyl transferases appeared to be regulated by both. Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP. Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities.

Show MeSH
Related in: MedlinePlus