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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, pCMV-Int or pC31-Intopt and pEGFPLuc were performed in A549 and Jurkat cell lines. Activity of φC31 integrase is expressed as β-galactosidase activity per milligram protein. The mean ± standard deviation of ratio of β-gal per protein (n = 5) is shown at 24 and 72 h post-transfection. (B) pDNA was isolated from A549 and Jurkat cells transfected with pSVpaxattP50-attB53 and pCMV-Int, pC31-Intopt 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. Jurkat cells co-transfected with pCMV-Int, pC31-Intopt, pUC21, respectively; A549 cells co-transfected with pCMV-Int, pC31-Intopt, pUC21, respectively; H2O. Strong bands could be detected for A549 cells by gel electrophoresis, whereas only weak bands could be detected for Jurkat cell line transfected with the integrase plasmids. pUC21 negative controls show no bands in any cell type.
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Figure 5: (A) Triple transfections of pSVpaxattP50-attB53, pCMV-Int or pC31-Intopt and pEGFPLuc were performed in A549 and Jurkat cell lines. Activity of φC31 integrase is expressed as β-galactosidase activity per milligram protein. The mean ± standard deviation of ratio of β-gal per protein (n = 5) is shown at 24 and 72 h post-transfection. (B) pDNA was isolated from A549 and Jurkat cells transfected with pSVpaxattP50-attB53 and pCMV-Int, pC31-Intopt 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. Jurkat cells co-transfected with pCMV-Int, pC31-Intopt, pUC21, respectively; A549 cells co-transfected with pCMV-Int, pC31-Intopt, pUC21, respectively; H2O. Strong bands could be detected for A549 cells by gel electrophoresis, whereas only weak bands could be detected for Jurkat cell line transfected with the integrase plasmids. pUC21 negative controls show no bands in any cell type.

Mentions: Twenty-four and 72 h after transfection with pC31-Intopt, β-galactosidase activity from plasmid A was 45- and 265-fold higher in A549 than in Jurkat cells, respectively. After transfection of A549 cells with pCMV-Int, β-galactosidase activity was 3- and 34-fold lower than after transfection with pC31-Intopt 24 and 72 h post-transfection, respectively. Transfection of Jurkat cells with pCMV-Int did not result in measurable β-galactosidase activity from plasmid A (Figure 5A). No β-galactosidase activity was observed in the negative pUC21 controls. These observations demonstrate that episomal recombination can be principally observed by measurement of active transcription of the recombination products in haematopoietic Jurkat cells by using a codon-optimized φC31 integrase.Figure 5.


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, pCMV-Int or pC31-Intopt and pEGFPLuc were performed in A549 and Jurkat cell lines. Activity of φC31 integrase is expressed as β-galactosidase activity per milligram protein. The mean ± standard deviation of ratio of β-gal per protein (n = 5) is shown at 24 and 72 h post-transfection. (B) pDNA was isolated from A549 and Jurkat cells transfected with pSVpaxattP50-attB53 and pCMV-Int, pC31-Intopt 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. Jurkat cells co-transfected with pCMV-Int, pC31-Intopt, pUC21, respectively; A549 cells co-transfected with pCMV-Int, pC31-Intopt, pUC21, respectively; H2O. Strong bands could be detected for A549 cells by gel electrophoresis, whereas only weak bands could be detected for Jurkat cell line transfected with the integrase plasmids. pUC21 negative controls show no bands in any cell type.
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Related In: Results  -  Collection

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Figure 5: (A) Triple transfections of pSVpaxattP50-attB53, pCMV-Int or pC31-Intopt and pEGFPLuc were performed in A549 and Jurkat cell lines. Activity of φC31 integrase is expressed as β-galactosidase activity per milligram protein. The mean ± standard deviation of ratio of β-gal per protein (n = 5) is shown at 24 and 72 h post-transfection. (B) pDNA was isolated from A549 and Jurkat cells transfected with pSVpaxattP50-attB53 and pCMV-Int, pC31-Intopt 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. Jurkat cells co-transfected with pCMV-Int, pC31-Intopt, pUC21, respectively; A549 cells co-transfected with pCMV-Int, pC31-Intopt, pUC21, respectively; H2O. Strong bands could be detected for A549 cells by gel electrophoresis, whereas only weak bands could be detected for Jurkat cell line transfected with the integrase plasmids. pUC21 negative controls show no bands in any cell type.
Mentions: Twenty-four and 72 h after transfection with pC31-Intopt, β-galactosidase activity from plasmid A was 45- and 265-fold higher in A549 than in Jurkat cells, respectively. After transfection of A549 cells with pCMV-Int, β-galactosidase activity was 3- and 34-fold lower than after transfection with pC31-Intopt 24 and 72 h post-transfection, respectively. Transfection of Jurkat cells with pCMV-Int did not result in measurable β-galactosidase activity from plasmid A (Figure 5A). No β-galactosidase activity was observed in the negative pUC21 controls. These observations demonstrate that episomal recombination can be principally observed by measurement of active transcription of the recombination products in haematopoietic Jurkat cells by using a codon-optimized φC31 integrase.Figure 5.

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