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Cell type differences in activity of the Streptomyces bacteriophage phiC31 integrase.

Maucksch C, Aneja MK, Hennen E, Bohla A, Hoffmann F, Elfinger M, Rosenecker J, Rudolph C - Nucleic Acids Res. (2008)

Bottom Line: Up to 100-fold higher levels of recombination product was found in C31 integrase transfected A549 lung than Jurkat T cells.When the C31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found.As one possible inhibitor of the C31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of C31 integrase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Ludwig-Maximilians-University, 80337 Munich, Germany.

ABSTRACT
Genomic integration by the Streptomyces bacteriophage C31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the C31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34(+) haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of C31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stem cells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in C31 integrase transfected A549 lung than Jurkat T cells. When the C31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the C31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of C31 integrase activity.

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(A) Triple transfections of pSVpaxattP50-attB53, an integrase plasmid and pEGFPLuc were performed in various cell types. Activity of φC31 integrase is shown as β-galactosidase activity per milligram protein. The mean ± standard deviation of ratio of β-gal per protein (n = 5) is shown at 24 h post-transfection. (B) pDNA was isolated from cells transfected with pSVpaxattP50-attB53 and pdelCpG-Int or pUC21 control 24 h post-transfection. Amplification of a 0.94 kb fragment of the 1.1 kb recombined plasmid mediated by the functional φC31 integrase was carried out by PCR. A549, BEAS-2B, HepG2, HeLa, Jurkat cells co-transfected with pdelCpG-Int, respectively; A549, BEAS-2B, HepG2, HeLa, Jurkat cells co-transfected with negative control pUC21, respectively; H2O. Strong bands could be detected for all non-haematopoietic cell types transfected with the integrase plasmid by gel electrophoresis, whereas only a very weak band could be detected for Jurkat cell line. pUC21 negative controls show no bands in any cell type.
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Figure 4: (A) Triple transfections of pSVpaxattP50-attB53, an integrase plasmid and pEGFPLuc were performed in various cell types. Activity of φC31 integrase is shown as β-galactosidase activity per milligram protein. The mean ± standard deviation of ratio of β-gal per protein (n = 5) is shown at 24 h post-transfection. (B) pDNA was isolated from cells transfected with pSVpaxattP50-attB53 and pdelCpG-Int or pUC21 control 24 h post-transfection. Amplification of a 0.94 kb fragment of the 1.1 kb recombined plasmid mediated by the functional φC31 integrase was carried out by PCR. A549, BEAS-2B, HepG2, HeLa, Jurkat cells co-transfected with pdelCpG-Int, respectively; A549, BEAS-2B, HepG2, HeLa, Jurkat cells co-transfected with negative control pUC21, respectively; H2O. Strong bands could be detected for all non-haematopoietic cell types transfected with the integrase plasmid by gel electrophoresis, whereas only a very weak band could be detected for Jurkat cell line. pUC21 negative controls show no bands in any cell type.

Mentions: To specify whether the lack of integrase-mediated long-term transgene expression in T cells lines was only due to detection limits of luciferase or due to other yet unknown cell specific reasons, we set up an episomal recombination assay to investigate the φC31 integrase activity in more detail. Various cell types were co-transfected with pSVpaxattP50-attB53 and pdelCpG-Int or pCAG-Int together with an internal transfection control pEGFPLuc. The 7.2 kb pSVpaxattP50-attB53 comprises an ‘expression-blocking’ sequence flanked by attB and attP sites between the P1 promoter and the lacZ cDNA. This ‘blocking’ sequence prohibits expression of β-galactosidase. The φC31 integrase mediates the excision of the ‘blocking’ sequence and brings the promoter in frame with the lacZ gene. Successful recombination results in a 6.1 kb recombined plasmid A, comprising the P1 promoter in frame with the lacZ cDNA, which results in β-galactosidase expression after transcription, and in a 1.1 kb recombined plasmid B, comprising the ‘blocking’ sequence. For all experiments a negative control was performed using pUC21 instead of the integrase plasmid. By subtracting the pUC21 negative control background from the β-galactosidase activity of the replicates, which were transfected with the integrase pDNA, it is possible to calculate the relative recombination efficiency of the φC31 integrase in different cell types in terms of active transcription of the recombination product (plasmid A). Luciferase activity in all experiments confirmed successful transfections. β-galactosidase activity derived from plasmid A in all non-haematopoietic cell lines indicated functionality of the φC31 integrase, whereas no β-galactosidase activity was measured in any of the haematopoietic cell types (Figure 4A). The β-galactosidase expression was highest in bronchial BEAS-2B cells, intermediate in liver HepG2 and HeLa cervix carcinoma cells and lowest in alveolar type II A549 and huMSC cells, which suggests a cell type-specific activity of the φC31 integrase. To exclude effects due to variations of the transfection efficiency, different transfection methods and integrase constructs were investigated. A549 cells were co-transfected with pSVpaxattP50-attB53, pEGFPLuc and pdelCpG-Int, pCAG-Int(NLS) or pUC21, respectively, using either nucleofection, Metafectene Pro or Lipofectamin 2000. Although the transfection methods resulted in variations of luciferase expressions ranging from low to high levels, β-galactosidase activity derived from plasmid A could be measured for pdelCpG-Int and pCAG-Int(NLS) and correlated with luciferase expression, in contrast to pUC21 control (data not shown). Therefore, the assay is independent of the transfection method and the used integrase construct.Figure 4.


Cell type differences in activity of the Streptomyces bacteriophage phiC31 integrase.

Maucksch C, Aneja MK, Hennen E, Bohla A, Hoffmann F, Elfinger M, Rosenecker J, Rudolph C - Nucleic Acids Res. (2008)

(A) Triple transfections of pSVpaxattP50-attB53, an integrase plasmid and pEGFPLuc were performed in various cell types. Activity of φC31 integrase is shown as β-galactosidase activity per milligram protein. The mean ± standard deviation of ratio of β-gal per protein (n = 5) is shown at 24 h post-transfection. (B) pDNA was isolated from cells transfected with pSVpaxattP50-attB53 and pdelCpG-Int or pUC21 control 24 h post-transfection. Amplification of a 0.94 kb fragment of the 1.1 kb recombined plasmid mediated by the functional φC31 integrase was carried out by PCR. A549, BEAS-2B, HepG2, HeLa, Jurkat cells co-transfected with pdelCpG-Int, respectively; A549, BEAS-2B, HepG2, HeLa, Jurkat cells co-transfected with negative control pUC21, respectively; H2O. Strong bands could be detected for all non-haematopoietic cell types transfected with the integrase plasmid by gel electrophoresis, whereas only a very weak band could be detected for Jurkat cell line. pUC21 negative controls show no bands in any cell type.
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Related In: Results  -  Collection

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Figure 4: (A) Triple transfections of pSVpaxattP50-attB53, an integrase plasmid and pEGFPLuc were performed in various cell types. Activity of φC31 integrase is shown as β-galactosidase activity per milligram protein. The mean ± standard deviation of ratio of β-gal per protein (n = 5) is shown at 24 h post-transfection. (B) pDNA was isolated from cells transfected with pSVpaxattP50-attB53 and pdelCpG-Int or pUC21 control 24 h post-transfection. Amplification of a 0.94 kb fragment of the 1.1 kb recombined plasmid mediated by the functional φC31 integrase was carried out by PCR. A549, BEAS-2B, HepG2, HeLa, Jurkat cells co-transfected with pdelCpG-Int, respectively; A549, BEAS-2B, HepG2, HeLa, Jurkat cells co-transfected with negative control pUC21, respectively; H2O. Strong bands could be detected for all non-haematopoietic cell types transfected with the integrase plasmid by gel electrophoresis, whereas only a very weak band could be detected for Jurkat cell line. pUC21 negative controls show no bands in any cell type.
Mentions: To specify whether the lack of integrase-mediated long-term transgene expression in T cells lines was only due to detection limits of luciferase or due to other yet unknown cell specific reasons, we set up an episomal recombination assay to investigate the φC31 integrase activity in more detail. Various cell types were co-transfected with pSVpaxattP50-attB53 and pdelCpG-Int or pCAG-Int together with an internal transfection control pEGFPLuc. The 7.2 kb pSVpaxattP50-attB53 comprises an ‘expression-blocking’ sequence flanked by attB and attP sites between the P1 promoter and the lacZ cDNA. This ‘blocking’ sequence prohibits expression of β-galactosidase. The φC31 integrase mediates the excision of the ‘blocking’ sequence and brings the promoter in frame with the lacZ gene. Successful recombination results in a 6.1 kb recombined plasmid A, comprising the P1 promoter in frame with the lacZ cDNA, which results in β-galactosidase expression after transcription, and in a 1.1 kb recombined plasmid B, comprising the ‘blocking’ sequence. For all experiments a negative control was performed using pUC21 instead of the integrase plasmid. By subtracting the pUC21 negative control background from the β-galactosidase activity of the replicates, which were transfected with the integrase pDNA, it is possible to calculate the relative recombination efficiency of the φC31 integrase in different cell types in terms of active transcription of the recombination product (plasmid A). Luciferase activity in all experiments confirmed successful transfections. β-galactosidase activity derived from plasmid A in all non-haematopoietic cell lines indicated functionality of the φC31 integrase, whereas no β-galactosidase activity was measured in any of the haematopoietic cell types (Figure 4A). The β-galactosidase expression was highest in bronchial BEAS-2B cells, intermediate in liver HepG2 and HeLa cervix carcinoma cells and lowest in alveolar type II A549 and huMSC cells, which suggests a cell type-specific activity of the φC31 integrase. To exclude effects due to variations of the transfection efficiency, different transfection methods and integrase constructs were investigated. A549 cells were co-transfected with pSVpaxattP50-attB53, pEGFPLuc and pdelCpG-Int, pCAG-Int(NLS) or pUC21, respectively, using either nucleofection, Metafectene Pro or Lipofectamin 2000. Although the transfection methods resulted in variations of luciferase expressions ranging from low to high levels, β-galactosidase activity derived from plasmid A could be measured for pdelCpG-Int and pCAG-Int(NLS) and correlated with luciferase expression, in contrast to pUC21 control (data not shown). Therefore, the assay is independent of the transfection method and the used integrase construct.Figure 4.

Bottom Line: Up to 100-fold higher levels of recombination product was found in C31 integrase transfected A549 lung than Jurkat T cells.When the C31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found.As one possible inhibitor of the C31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of C31 integrase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Ludwig-Maximilians-University, 80337 Munich, Germany.

ABSTRACT
Genomic integration by the Streptomyces bacteriophage C31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the C31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34(+) haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of C31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stem cells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in C31 integrase transfected A549 lung than Jurkat T cells. When the C31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the C31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of C31 integrase activity.

Show MeSH
Related in: MedlinePlus