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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) Co-transfections of 1 µg pdelCpG-Luc-attB and 4 µg integrase/control plasmids, respectively, were performed in Jurkat cell line. No significant long-term enhancement of luciferase activity could be detected for the integrase constructs above pdelCpG control. (B) Co-transfections of 1 µg pdelCpG-Luc-attB and various amounts of mRNA coding for φC31 integrase, respectively, were performed in Jurkat cell line. Significant enhancement of luciferase activity above control could be detected at two time points for 2 µg of integrase mRNA. (C) Co-transfections of 1 µg pdelCpG-Luc-attB and various amounts of mRNA or different plasmids coding for φC31 integrase, respectively, were performed in ED-7R cell line. No luciferase activity could be detected for any construct after day 10. Nucleofection was used for all transfections. Luciferase values are relative to day 1 values. All experiments were performed in five replicates (n = 5).
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Figure 2: (A) Co-transfections of 1 µg pdelCpG-Luc-attB and 4 µg integrase/control plasmids, respectively, were performed in Jurkat cell line. No significant long-term enhancement of luciferase activity could be detected for the integrase constructs above pdelCpG control. (B) Co-transfections of 1 µg pdelCpG-Luc-attB and various amounts of mRNA coding for φC31 integrase, respectively, were performed in Jurkat cell line. Significant enhancement of luciferase activity above control could be detected at two time points for 2 µg of integrase mRNA. (C) Co-transfections of 1 µg pdelCpG-Luc-attB and various amounts of mRNA or different plasmids coding for φC31 integrase, respectively, were performed in ED-7R cell line. No luciferase activity could be detected for any construct after day 10. Nucleofection was used for all transfections. Luciferase values are relative to day 1 values. All experiments were performed in five replicates (n = 5).

Mentions: Jurkat T cells were co-transfected with different constructs coding for the φC31 integrase and pdelCpG-Luc-attB, containing the attB sequence and a luciferase gene in a CpG dinucleotide reduced backbone. The following integrase constructs were tested: pCMV-Int, pdelCpG-Int, pVAX1-Int, pPGK-Int(NLS) and pCAG-Int(NLS) (Figure 1). pdelCpG was used as a negative control for the integrase plasmids. These plasmids were co-transfected with pdelCpG-Luc-attB in a ratio of 4:1 (w/w) using nucleofection, which is an electroporation-based transfection method and has previously been shown to efficiently transfect suspension cells (17–19). Co-transfection of pDNA coding for the φC31 integrase compared to the negative control pdelCpG (Figure 2A) or using pdelCpG-Luc lacking attB could not significantly enhance long-term luciferase expression in Jurkat cells (data not shown). One of the reasons for the lack of long-term gene expression may have been low integrase expression levels. As it has previously been shown that mRNA transfection in haematopoietic monocytes is more efficient than pDNA transfections (20), additional experiments were performed using φC31 integrase mRNA instead of pDNA. Using nucleofection, 0.5, 1.0 or 2.0 µg of integrase mRNA was co-transfected with 1 µg pdelCpG-Luc-attB. pdelCpG vector was used as negative control in these experiments. With integrase mRNA, significantly enhanced long-term gene expression, compared to the negative control, could be observed up to day 33 post-transfection when a high amount (2 µg) of integrase mRNA was used (Figure 2B). We repeated these experiments in another T cell line ED-7R, but in contrast to Jurkat cells, transgene expression was not significantly different from pdelCpG control transfections (Figure 2C). Similar results were obtained when these long-term expression experiments were repeated using electroporation instead of nucleofection as gene delivery method, using pEGFPLucattB instead of pdelCpG-Luc-attB or using different ratios of integrase plasmids to luc-attB plasmid (data not shown).Figure 2.


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) Co-transfections of 1 µg pdelCpG-Luc-attB and 4 µg integrase/control plasmids, respectively, were performed in Jurkat cell line. No significant long-term enhancement of luciferase activity could be detected for the integrase constructs above pdelCpG control. (B) Co-transfections of 1 µg pdelCpG-Luc-attB and various amounts of mRNA coding for φC31 integrase, respectively, were performed in Jurkat cell line. Significant enhancement of luciferase activity above control could be detected at two time points for 2 µg of integrase mRNA. (C) Co-transfections of 1 µg pdelCpG-Luc-attB and various amounts of mRNA or different plasmids coding for φC31 integrase, respectively, were performed in ED-7R cell line. No luciferase activity could be detected for any construct after day 10. Nucleofection was used for all transfections. Luciferase values are relative to day 1 values. All experiments were performed in five replicates (n = 5).
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Figure 2: (A) Co-transfections of 1 µg pdelCpG-Luc-attB and 4 µg integrase/control plasmids, respectively, were performed in Jurkat cell line. No significant long-term enhancement of luciferase activity could be detected for the integrase constructs above pdelCpG control. (B) Co-transfections of 1 µg pdelCpG-Luc-attB and various amounts of mRNA coding for φC31 integrase, respectively, were performed in Jurkat cell line. Significant enhancement of luciferase activity above control could be detected at two time points for 2 µg of integrase mRNA. (C) Co-transfections of 1 µg pdelCpG-Luc-attB and various amounts of mRNA or different plasmids coding for φC31 integrase, respectively, were performed in ED-7R cell line. No luciferase activity could be detected for any construct after day 10. Nucleofection was used for all transfections. Luciferase values are relative to day 1 values. All experiments were performed in five replicates (n = 5).
Mentions: Jurkat T cells were co-transfected with different constructs coding for the φC31 integrase and pdelCpG-Luc-attB, containing the attB sequence and a luciferase gene in a CpG dinucleotide reduced backbone. The following integrase constructs were tested: pCMV-Int, pdelCpG-Int, pVAX1-Int, pPGK-Int(NLS) and pCAG-Int(NLS) (Figure 1). pdelCpG was used as a negative control for the integrase plasmids. These plasmids were co-transfected with pdelCpG-Luc-attB in a ratio of 4:1 (w/w) using nucleofection, which is an electroporation-based transfection method and has previously been shown to efficiently transfect suspension cells (17–19). Co-transfection of pDNA coding for the φC31 integrase compared to the negative control pdelCpG (Figure 2A) or using pdelCpG-Luc lacking attB could not significantly enhance long-term luciferase expression in Jurkat cells (data not shown). One of the reasons for the lack of long-term gene expression may have been low integrase expression levels. As it has previously been shown that mRNA transfection in haematopoietic monocytes is more efficient than pDNA transfections (20), additional experiments were performed using φC31 integrase mRNA instead of pDNA. Using nucleofection, 0.5, 1.0 or 2.0 µg of integrase mRNA was co-transfected with 1 µg pdelCpG-Luc-attB. pdelCpG vector was used as negative control in these experiments. With integrase mRNA, significantly enhanced long-term gene expression, compared to the negative control, could be observed up to day 33 post-transfection when a high amount (2 µg) of integrase mRNA was used (Figure 2B). We repeated these experiments in another T cell line ED-7R, but in contrast to Jurkat cells, transgene expression was not significantly different from pdelCpG control transfections (Figure 2C). Similar results were obtained when these long-term expression experiments were repeated using electroporation instead of nucleofection as gene delivery method, using pEGFPLucattB instead of pdelCpG-Luc-attB or using different ratios of integrase plasmids to luc-attB plasmid (data not shown).Figure 2.

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