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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) Cell lysates of either pCMV-Int transfected A549 and Jurkat cells or pC31-Intopt transfected A549 and Jurkat cells were analysed by western blotting. Cell lysates of untreated A549 and Jurkat cells are represented. Detection of the integrase protein positive control is shown. (B) Total RNA was isolated from A549 and Jurkat cells 24 h post-transfection with pSVpaxattP50-attB53 and either pCMV-Int or (C) pC31-Intopt. Total cDNA was produced by reverse transcriptase and integrase cDNA was quantified by qRT-PCR. The values represent the relative mRNA integrase expression normalized to pCMV-Int in Jurkat cells 24 h post-transfection.
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Figure 3: (A) Cell lysates of either pCMV-Int transfected A549 and Jurkat cells or pC31-Intopt transfected A549 and Jurkat cells were analysed by western blotting. Cell lysates of untreated A549 and Jurkat cells are represented. Detection of the integrase protein positive control is shown. (B) Total RNA was isolated from A549 and Jurkat cells 24 h post-transfection with pSVpaxattP50-attB53 and either pCMV-Int or (C) pC31-Intopt. Total cDNA was produced by reverse transcriptase and integrase cDNA was quantified by qRT-PCR. The values represent the relative mRNA integrase expression normalized to pCMV-Int in Jurkat cells 24 h post-transfection.

Mentions: The expression studies in Jurkat and ED-7R cells suggested either inefficient integrase expression or low recombination efficiency of the φC31 integrase to achieve long-term gene expression in T cell lines. To confirm the expression of the integrase protein in the experimental settings, Jurkat cells were transfected with either pCMV-Int or the codon-optimized pC31-Intopt and western blot analysis for the integrase protein was performed 24 h post-transfection. As positive control for the integrase expression constructs, A549 lung cells were transfected with the same plasmids, a cell line in which it has previously been shown that the integrase mediates long-term gene expression (10). Integrase specific bands for both constructs were detected in A549 and Jurkat T cells, respectively, and expression levels (either native or codon optimized) were equivalent in each cell line (Figure 3A). Further, integrase mRNA was quantified for pCMV-Int and pC31-Intopt using qRT-PCR in both cell lines, respectively. The pCMV-Int integrase mRNA level was significantly 5.8-fold higher in A549 cells than in Jurkat cells (Figure 3B), whereas no significant difference was detected for pC31-Intopt mRNA (Figure 3C).Figure 3.


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) Cell lysates of either pCMV-Int transfected A549 and Jurkat cells or pC31-Intopt transfected A549 and Jurkat cells were analysed by western blotting. Cell lysates of untreated A549 and Jurkat cells are represented. Detection of the integrase protein positive control is shown. (B) Total RNA was isolated from A549 and Jurkat cells 24 h post-transfection with pSVpaxattP50-attB53 and either pCMV-Int or (C) pC31-Intopt. Total cDNA was produced by reverse transcriptase and integrase cDNA was quantified by qRT-PCR. The values represent the relative mRNA integrase expression normalized to pCMV-Int in Jurkat cells 24 h post-transfection.
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Related In: Results  -  Collection

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Figure 3: (A) Cell lysates of either pCMV-Int transfected A549 and Jurkat cells or pC31-Intopt transfected A549 and Jurkat cells were analysed by western blotting. Cell lysates of untreated A549 and Jurkat cells are represented. Detection of the integrase protein positive control is shown. (B) Total RNA was isolated from A549 and Jurkat cells 24 h post-transfection with pSVpaxattP50-attB53 and either pCMV-Int or (C) pC31-Intopt. Total cDNA was produced by reverse transcriptase and integrase cDNA was quantified by qRT-PCR. The values represent the relative mRNA integrase expression normalized to pCMV-Int in Jurkat cells 24 h post-transfection.
Mentions: The expression studies in Jurkat and ED-7R cells suggested either inefficient integrase expression or low recombination efficiency of the φC31 integrase to achieve long-term gene expression in T cell lines. To confirm the expression of the integrase protein in the experimental settings, Jurkat cells were transfected with either pCMV-Int or the codon-optimized pC31-Intopt and western blot analysis for the integrase protein was performed 24 h post-transfection. As positive control for the integrase expression constructs, A549 lung cells were transfected with the same plasmids, a cell line in which it has previously been shown that the integrase mediates long-term gene expression (10). Integrase specific bands for both constructs were detected in A549 and Jurkat T cells, respectively, and expression levels (either native or codon optimized) were equivalent in each cell line (Figure 3A). Further, integrase mRNA was quantified for pCMV-Int and pC31-Intopt using qRT-PCR in both cell lines, respectively. The pCMV-Int integrase mRNA level was significantly 5.8-fold higher in A549 cells than in Jurkat cells (Figure 3B), whereas no significant difference was detected for pC31-Intopt mRNA (Figure 3C).Figure 3.

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