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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) DAXX protein was detected 24 and 72 h post-transfections of CMV-Int in A549 and Jurkat cells by western blotting, respectively. (B) Total RNA was isolated from A549 and Jurkat cells 24 and 72 h post-transfection with pSVpaxattP50-attB53 and pCMV-Int. Total cDNA was produced by reverse transcriptase and DAXX cDNA was quantified by qRT-PCR.
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Figure 7: (A) DAXX protein was detected 24 and 72 h post-transfections of CMV-Int in A549 and Jurkat cells by western blotting, respectively. (B) Total RNA was isolated from A549 and Jurkat cells 24 and 72 h post-transfection with pSVpaxattP50-attB53 and pCMV-Int. Total cDNA was produced by reverse transcriptase and DAXX cDNA was quantified by qRT-PCR.

Mentions: DAXX protein has been previously shown to interact with φC31 integrase and inhibit its recombination efficiency (14). To investigate if cell type specific differences in the amount of DAXX protein could be a likely reason for the reduced activity of the φC31 integrase in T cells, western blot analysis of DAXX protein in Jurkat and A549 cells was carried out. Indeed, higher amounts of DAXX protein were observed in Jurkat than in A549 cells by semi-quantification of the western blot analysis (Figure 7A). Further, qRT-PCR was performed to quantify DAXX on mRNA level in A549 and Jurkat cells. The amounts of DAXX were significantly 2.7- and 5.4-fold higher in Jurkat than in A549 cells 24 and 72 h post-transfection, respectively (Figure 7B).Figure 7.


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) DAXX protein was detected 24 and 72 h post-transfections of CMV-Int in A549 and Jurkat cells by western blotting, respectively. (B) Total RNA was isolated from A549 and Jurkat cells 24 and 72 h post-transfection with pSVpaxattP50-attB53 and pCMV-Int. Total cDNA was produced by reverse transcriptase and DAXX cDNA was quantified by qRT-PCR.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553569&req=5

Figure 7: (A) DAXX protein was detected 24 and 72 h post-transfections of CMV-Int in A549 and Jurkat cells by western blotting, respectively. (B) Total RNA was isolated from A549 and Jurkat cells 24 and 72 h post-transfection with pSVpaxattP50-attB53 and pCMV-Int. Total cDNA was produced by reverse transcriptase and DAXX cDNA was quantified by qRT-PCR.
Mentions: DAXX protein has been previously shown to interact with φC31 integrase and inhibit its recombination efficiency (14). To investigate if cell type specific differences in the amount of DAXX protein could be a likely reason for the reduced activity of the φC31 integrase in T cells, western blot analysis of DAXX protein in Jurkat and A549 cells was carried out. Indeed, higher amounts of DAXX protein were observed in Jurkat than in A549 cells by semi-quantification of the western blot analysis (Figure 7A). Further, qRT-PCR was performed to quantify DAXX on mRNA level in A549 and Jurkat cells. The amounts of DAXX were significantly 2.7- and 5.4-fold higher in Jurkat than in A549 cells 24 and 72 h post-transfection, respectively (Figure 7B).Figure 7.

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