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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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pDNA was isolated from A549 and Jurkat cells transfected with pSVpaxattP50-attB53 and pCMV-Int, pC31-Intopt or pUC21 control 24 and 72 h post-transfection was amplified and quantified by qRT-PCR. The amount of recombination product Plasmid B of the φC31 integrase is shown for A549 and Jurkat cells 24 and 72 h post-transfection. The values represent the amounts of recombination products normalized to pCMV-Int in Jurkat cells 24 h post-transfection.
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Figure 6: pDNA was isolated from A549 and Jurkat cells transfected with pSVpaxattP50-attB53 and pCMV-Int, pC31-Intopt or pUC21 control 24 and 72 h post-transfection was amplified and quantified by qRT-PCR. The amount of recombination product Plasmid B of the φC31 integrase is shown for A549 and Jurkat cells 24 and 72 h post-transfection. The values represent the amounts of recombination products normalized to pCMV-Int in Jurkat cells 24 h post-transfection.

Mentions: However, in order to directly investigate episomal recombination, the recombination products were further analysed by PCR and qRT-PCR. A strong band of the expected 0.94 kb fragment of the recombination product plasmid B was detected in A549 cells after transfection with pCMV-Int and pC31-Intopt, whereas only a weak band was observed in Jurkat cells 24 h post-transfection (Figure 5B). Twenty-four hours after transfection, qRT-PCR resulted in statistically significant 100- and 29-fold higher amounts of recombination product plasmid B in A549 than in Jurkat cells for pCMV-Int and pC31-Intopt, respectively. After 72 h, 3- and 120-fold higher amounts of recombination products were detected in A549 than in Jurkat cells for pCMV-Int and pC31-Intopt, respectively (Figure 6). Together with the measured mRNA levels after transfection with pCMV-Int or pC31-Intopt (Figure 3B and C), the φC31 integrase recombination efficacy can be normalized to the integrase mRNA levels for each of the cell lines by dividing the amount of recombination product plasmid B by the amount of integrase mRNA (Table 1). In independent experiments with either pCMV-Int or pC31-Intopt, a 17- and 16-fold higher normalized φC31 integrase activity was observed in A549 than in Jurkat cells 24 h post-transfection, respectively (Table 2). These data demonstrate that the φC31 integrase recombination efficacy is markedly reduced in haematopoietic Jurkat T cells compared with lung cells.Figure 6.


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)

pDNA was isolated from A549 and Jurkat cells transfected with pSVpaxattP50-attB53 and pCMV-Int, pC31-Intopt or pUC21 control 24 and 72 h post-transfection was amplified and quantified by qRT-PCR. The amount of recombination product Plasmid B of the φC31 integrase is shown for A549 and Jurkat cells 24 and 72 h post-transfection. The values represent the amounts of recombination products normalized to pCMV-Int in Jurkat cells 24 h post-transfection.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553569&req=5

Figure 6: pDNA was isolated from A549 and Jurkat cells transfected with pSVpaxattP50-attB53 and pCMV-Int, pC31-Intopt or pUC21 control 24 and 72 h post-transfection was amplified and quantified by qRT-PCR. The amount of recombination product Plasmid B of the φC31 integrase is shown for A549 and Jurkat cells 24 and 72 h post-transfection. The values represent the amounts of recombination products normalized to pCMV-Int in Jurkat cells 24 h post-transfection.
Mentions: However, in order to directly investigate episomal recombination, the recombination products were further analysed by PCR and qRT-PCR. A strong band of the expected 0.94 kb fragment of the recombination product plasmid B was detected in A549 cells after transfection with pCMV-Int and pC31-Intopt, whereas only a weak band was observed in Jurkat cells 24 h post-transfection (Figure 5B). Twenty-four hours after transfection, qRT-PCR resulted in statistically significant 100- and 29-fold higher amounts of recombination product plasmid B in A549 than in Jurkat cells for pCMV-Int and pC31-Intopt, respectively. After 72 h, 3- and 120-fold higher amounts of recombination products were detected in A549 than in Jurkat cells for pCMV-Int and pC31-Intopt, respectively (Figure 6). Together with the measured mRNA levels after transfection with pCMV-Int or pC31-Intopt (Figure 3B and C), the φC31 integrase recombination efficacy can be normalized to the integrase mRNA levels for each of the cell lines by dividing the amount of recombination product plasmid B by the amount of integrase mRNA (Table 1). In independent experiments with either pCMV-Int or pC31-Intopt, a 17- and 16-fold higher normalized φC31 integrase activity was observed in A549 than in Jurkat cells 24 h post-transfection, respectively (Table 2). These data demonstrate that the φC31 integrase recombination efficacy is markedly reduced in haematopoietic Jurkat T cells compared with lung cells.Figure 6.

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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Related in: MedlinePlus