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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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Schematic representation of the pDNA constructs used in the present study.
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Figure 1: Schematic representation of the pDNA constructs used in the present study.

Mentions: pCMVInt has been previously described (8,9,12) and was kindly provided by M. P. Calos (Stanford University, USA). pCAG-Int(NLS) and pPGK-Int(NLS) coding for an integrase fused with a C-terminal nuclear localization signal (NLS) of SV40 T antigen, driven by CAG and PGK promoters, respectively, and pSVpaxattP50-attB53, comprising an ‘expression blocking’ sequence flanked by attB and attP sites between a P1 promoter and a lacZ gene, were kindly provided by R. Kuehn (Helmholtz Zentrum Munich, German Research Centre for Environmental Health, Germany). pC31-Intopt was obtained from Addgene Inc. (Cambridge, MA, USA). pVAX1-Int was constructed by excising the integrase cDNA from pCMV-Int by PstI-XhoI (Fermentas, St. Leon-Rot, Germany) digestion and cloning into respective sites of pVAX1 (Invitrogen, Paisley, UK). pdelCpG-GFP was obtained from Geneart (GENEART AG, Regensburg, Germany) and contains the same expression cassette as pVAX1 but has reduced number of CpG motifs. The GFP expression cassette was removed from this plasmid by HindIII–BamHI digestion and self-circularized to obtain pdelCpG. Fragment containing the integrase was excised from pVAX1-Int by PmeI digestion and cloned into PmeI site of pdelCpG to generate pdelCpG-Int. mRNA coding for the φC31 integrase was produced by CureVac (CUREVAC GmbH, Tuebingen, Germany). pVAX1-Luc was constructed as a shuttle vector by excising firefly luciferase cDNA from pGL3-Basic vector (Promega, Madison, WI, USA) by HindIII–XbaI digestion and cloning into the corresponding sites of pVAX1. A HindIII–PmeI fragment from pVAX1-Luc was then cloned into the same sites of pdelCpG-GFP, thus replacing GFP with luciferase and generating pdelCpG-Luc. φC31 integrase recognition sequence attB was excised from pTA-attB (provided by M. P. Calos) by EcoRI digestion and was cloned blunt ended into the BglII site of pdelCpG-Luc to obtain pdelCpG-Luc-attB. pEGFPLucattB was kindly provided by T. W. Chalberg (Stanford University, USA). Briefly, the plasmid codes for the fusion protein of EGFP and luciferase driven by the CMV promoter and contains the attB recognition site for the integrase in the plasmid backbone adjacent to the ampicillin selection marker. All constructs used in this study are schematically represented in Figure 1.Figure 1.


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)

Schematic representation of the pDNA constructs used in the present study.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Schematic representation of the pDNA constructs used in the present study.
Mentions: pCMVInt has been previously described (8,9,12) and was kindly provided by M. P. Calos (Stanford University, USA). pCAG-Int(NLS) and pPGK-Int(NLS) coding for an integrase fused with a C-terminal nuclear localization signal (NLS) of SV40 T antigen, driven by CAG and PGK promoters, respectively, and pSVpaxattP50-attB53, comprising an ‘expression blocking’ sequence flanked by attB and attP sites between a P1 promoter and a lacZ gene, were kindly provided by R. Kuehn (Helmholtz Zentrum Munich, German Research Centre for Environmental Health, Germany). pC31-Intopt was obtained from Addgene Inc. (Cambridge, MA, USA). pVAX1-Int was constructed by excising the integrase cDNA from pCMV-Int by PstI-XhoI (Fermentas, St. Leon-Rot, Germany) digestion and cloning into respective sites of pVAX1 (Invitrogen, Paisley, UK). pdelCpG-GFP was obtained from Geneart (GENEART AG, Regensburg, Germany) and contains the same expression cassette as pVAX1 but has reduced number of CpG motifs. The GFP expression cassette was removed from this plasmid by HindIII–BamHI digestion and self-circularized to obtain pdelCpG. Fragment containing the integrase was excised from pVAX1-Int by PmeI digestion and cloned into PmeI site of pdelCpG to generate pdelCpG-Int. mRNA coding for the φC31 integrase was produced by CureVac (CUREVAC GmbH, Tuebingen, Germany). pVAX1-Luc was constructed as a shuttle vector by excising firefly luciferase cDNA from pGL3-Basic vector (Promega, Madison, WI, USA) by HindIII–XbaI digestion and cloning into the corresponding sites of pVAX1. A HindIII–PmeI fragment from pVAX1-Luc was then cloned into the same sites of pdelCpG-GFP, thus replacing GFP with luciferase and generating pdelCpG-Luc. φC31 integrase recognition sequence attB was excised from pTA-attB (provided by M. P. Calos) by EcoRI digestion and was cloned blunt ended into the BglII site of pdelCpG-Luc to obtain pdelCpG-Luc-attB. pEGFPLucattB was kindly provided by T. W. Chalberg (Stanford University, USA). Briefly, the plasmid codes for the fusion protein of EGFP and luciferase driven by the CMV promoter and contains the attB recognition site for the integrase in the plasmid backbone adjacent to the ampicillin selection marker. All constructs used in this study are schematically represented in Figure 1.Figure 1.

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