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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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Pax6 expressing FlpIn-3T3 cells have higher proliferation and higher migration capacity compared to FlpIn-3T3 control cells and Pax6(5a) expressing cells.Control, Pax6 and Pax6(5a) cells were seeded in xCELLigence (Roche) proliferation plates, cells were left on the bench for 1 hr before the plates were put in the xCELLigence machine for measurements for 24 hrs. The measured values were used to calculate the (A) adhesion slope, (B) proliferation slope and (C) doubling time. For adhesion and proliferation slope analyses the mean and standard deviation of three independent experiments are shown. The doubling time analysis shows one representative experiment out of three, because absolute values were changing from experiment to experiment probably due to fluctuations in cell condition. However, described pattern of increased proliferation (Pax6>Pax6(5a)>control) was consistent in all experiments. Each experiment was done in triplicates (A–C). xCELLigence migration plates were used to measure the migration, and to calculate the migration slope (D). One representative experiment out of 3 is shown.
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pone-0031915-g008: Pax6 expressing FlpIn-3T3 cells have higher proliferation and higher migration capacity compared to FlpIn-3T3 control cells and Pax6(5a) expressing cells.Control, Pax6 and Pax6(5a) cells were seeded in xCELLigence (Roche) proliferation plates, cells were left on the bench for 1 hr before the plates were put in the xCELLigence machine for measurements for 24 hrs. The measured values were used to calculate the (A) adhesion slope, (B) proliferation slope and (C) doubling time. For adhesion and proliferation slope analyses the mean and standard deviation of three independent experiments are shown. The doubling time analysis shows one representative experiment out of three, because absolute values were changing from experiment to experiment probably due to fluctuations in cell condition. However, described pattern of increased proliferation (Pax6>Pax6(5a)>control) was consistent in all experiments. Each experiment was done in triplicates (A–C). xCELLigence migration plates were used to measure the migration, and to calculate the migration slope (D). One representative experiment out of 3 is shown.

Mentions: Consistent with the fact that about half of the target genes identified by the gene expression microarray are associated with the membrane and extracellular region, there is a change in morphology from the FlpIn-3T3 parent cell line to the Pax6 and Pax6(5a) cell lines. The Pax6 and Pax6(5a) cells are bigger and more flattened, and seemingly have more protrusions (Fig. 7). In order to evaluate if Pax6 or Pax6(5a) can influence major biological processes in 3T3 cells, we employed a recently introduced xCELLigence platform (Roche) which facilitates studying of cell proliferation and migration in real time. This is a microelectric assay based on changing impedance of electrodes in presence of cells. We recognize that differences in morphology of the three types of 3T3 cells studied (control, Pax6 and Pax6(5a)) could make impedance-derived absolute values misleading. Therefore the data are presented as curve slopes or doubling times. Attachment and initial spreading of the cells typically took 3±1 hours, and this time interval was chosen for calculation of attachment slopes as depicted on figure 8A. We observed repeatedly that 3T3-Pax6 cells attach more rapidly than 3T3-control cells, while no or little difference was seen for 3T3-Pax6(5a) cells compared to the control. In all experiments (n = 4) the 3T3 cell lines expressing either of isoforms of Pax6 had higher proliferation rates compared to the control cells, with 3T3-Pax6 being most prominent. This is shown both by proliferation slope analysis (Fig. 8B) and doubling time calculation (Fig. 8C). Finally, we studied whether serum starved 3T3-Pax6 or 3T3-Pax(5a) cells had altered migration capabilities in presence of a serum attractant compared to the 3T3-control cells. 3T3-control cells were unable to migrate through the uncoated xCELLigence CIM-plate membrane in any of the three performed experiments. To minimize the possibility that the observed difference in migration could be due to faster proliferating Pax6 cells the experiment was limited to 24 hrs. Furtermore, cells attached to the membrane at the end of the experiment were stained, and confirmed several fold differences in cell numbers (data not shown), ruling out the possibility that the observed changes in impedance were solely caused by adherence differences. Interestingly, Pax6 expressing cells did actively pass through the membrane pores following the serum gradient, while Pax6(5a) seemed to have very limited influence on the migration abilities of the 3T3 cells (Fig. 8D).


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)

Pax6 expressing FlpIn-3T3 cells have higher proliferation and higher migration capacity compared to FlpIn-3T3 control cells and Pax6(5a) expressing cells.Control, Pax6 and Pax6(5a) cells were seeded in xCELLigence (Roche) proliferation plates, cells were left on the bench for 1 hr before the plates were put in the xCELLigence machine for measurements for 24 hrs. The measured values were used to calculate the (A) adhesion slope, (B) proliferation slope and (C) doubling time. For adhesion and proliferation slope analyses the mean and standard deviation of three independent experiments are shown. The doubling time analysis shows one representative experiment out of three, because absolute values were changing from experiment to experiment probably due to fluctuations in cell condition. However, described pattern of increased proliferation (Pax6>Pax6(5a)>control) was consistent in all experiments. Each experiment was done in triplicates (A–C). xCELLigence migration plates were used to measure the migration, and to calculate the migration slope (D). One representative experiment out of 3 is shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285655&req=5

pone-0031915-g008: Pax6 expressing FlpIn-3T3 cells have higher proliferation and higher migration capacity compared to FlpIn-3T3 control cells and Pax6(5a) expressing cells.Control, Pax6 and Pax6(5a) cells were seeded in xCELLigence (Roche) proliferation plates, cells were left on the bench for 1 hr before the plates were put in the xCELLigence machine for measurements for 24 hrs. The measured values were used to calculate the (A) adhesion slope, (B) proliferation slope and (C) doubling time. For adhesion and proliferation slope analyses the mean and standard deviation of three independent experiments are shown. The doubling time analysis shows one representative experiment out of three, because absolute values were changing from experiment to experiment probably due to fluctuations in cell condition. However, described pattern of increased proliferation (Pax6>Pax6(5a)>control) was consistent in all experiments. Each experiment was done in triplicates (A–C). xCELLigence migration plates were used to measure the migration, and to calculate the migration slope (D). One representative experiment out of 3 is shown.
Mentions: Consistent with the fact that about half of the target genes identified by the gene expression microarray are associated with the membrane and extracellular region, there is a change in morphology from the FlpIn-3T3 parent cell line to the Pax6 and Pax6(5a) cell lines. The Pax6 and Pax6(5a) cells are bigger and more flattened, and seemingly have more protrusions (Fig. 7). In order to evaluate if Pax6 or Pax6(5a) can influence major biological processes in 3T3 cells, we employed a recently introduced xCELLigence platform (Roche) which facilitates studying of cell proliferation and migration in real time. This is a microelectric assay based on changing impedance of electrodes in presence of cells. We recognize that differences in morphology of the three types of 3T3 cells studied (control, Pax6 and Pax6(5a)) could make impedance-derived absolute values misleading. Therefore the data are presented as curve slopes or doubling times. Attachment and initial spreading of the cells typically took 3±1 hours, and this time interval was chosen for calculation of attachment slopes as depicted on figure 8A. We observed repeatedly that 3T3-Pax6 cells attach more rapidly than 3T3-control cells, while no or little difference was seen for 3T3-Pax6(5a) cells compared to the control. In all experiments (n = 4) the 3T3 cell lines expressing either of isoforms of Pax6 had higher proliferation rates compared to the control cells, with 3T3-Pax6 being most prominent. This is shown both by proliferation slope analysis (Fig. 8B) and doubling time calculation (Fig. 8C). Finally, we studied whether serum starved 3T3-Pax6 or 3T3-Pax(5a) cells had altered migration capabilities in presence of a serum attractant compared to the 3T3-control cells. 3T3-control cells were unable to migrate through the uncoated xCELLigence CIM-plate membrane in any of the three performed experiments. To minimize the possibility that the observed difference in migration could be due to faster proliferating Pax6 cells the experiment was limited to 24 hrs. Furtermore, cells attached to the membrane at the end of the experiment were stained, and confirmed several fold differences in cell numbers (data not shown), ruling out the possibility that the observed changes in impedance were solely caused by adherence differences. Interestingly, Pax6 expressing cells did actively pass through the membrane pores following the serum gradient, while Pax6(5a) seemed to have very limited influence on the migration abilities of the 3T3 cells (Fig. 8D).

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