Irina Hellgren1, Julianne Gorman, Christer Sylvén. 1. Karolinska Institute at Department of Cardiology at Huddinge University Hospital, Stockholm, Sweden. irina.hellgren@medhs.ki.se
Abstract
BACKGROUND: The polycationic nanopeptide RKKRRQRRR is a fragment of HIV-Tat protein known to be a transfection enhancer. Such factors control the transfection efficiency of plasmid DNA complexed with tat peptide. However, their actions are poorly understood. METHODS AND RESULTS: Several cell lines and primary cells were used in transfection experiments. The optimal charge ratio of plasmid to the branched 8Tat peptide was 1:8 for all cell lines and primary cells tested except primary human skin fibroblasts where it was 1:4. The conditions of complex formation (volume, DNA-peptide-solution mixing order and the nature of solution (HEPES, 0.15 M NaCl or 5% dextrose)) influenced transfection efficiency. Chloroquine increased the transgene expression 50-fold in HeLa cells but had little impact on Cos7 cells or 3T3 fibroblasts. The integrity of plasmid DNA in the presence of DNase I was shown to be dependent on the DNA/Tat ratio and the nature of the complex solvent. Complexes with -/+ charge ratio of 1:8 formulated in NaCl and dextrose provided high degree of DNA resistance towards nuclease degradation. CONCLUSIONS: The conditions of DNA-peptide complex formation and DNA/Tat ratio have significant impact on the level of transgene expression and degree of DNA protection from nuclease attack. The level of elevation of gene delivery in the presence of chloroquine varied considerably between different cells, indicating possible different mechanisms of plasmid-peptide internalisation and intracellular trafficking. The efficiency of branched 8Tat peptide as a plasmid DNA carrier varied between different cell types and have to be optimised for each application.
BACKGROUND: The polycationic nanopeptide RKKRRQRRR is a fragment of HIV-Tat protein known to be a transfection enhancer. Such factors control the transfection efficiency of plasmid DNA complexed with tat peptide. However, their actions are poorly understood. METHODS AND RESULTS: Several cell lines and primary cells were used in transfection experiments. The optimal charge ratio of plasmid to the branched 8Tat peptide was 1:8 for all cell lines and primary cells tested except primary human skin fibroblasts where it was 1:4. The conditions of complex formation (volume, DNA-peptide-solution mixing order and the nature of solution (HEPES, 0.15 M NaCl or 5% dextrose)) influenced transfection efficiency. Chloroquine increased the transgene expression 50-fold in HeLa cells but had little impact on Cos7 cells or 3T3 fibroblasts. The integrity of plasmid DNA in the presence of DNase I was shown to be dependent on the DNA/Tat ratio and the nature of the complex solvent. Complexes with -/+ charge ratio of 1:8 formulated in NaCl and dextrose provided high degree of DNA resistance towards nuclease degradation. CONCLUSIONS: The conditions of DNA-peptide complex formation and DNA/Tat ratio have significant impact on the level of transgene expression and degree of DNA protection from nuclease attack. The level of elevation of gene delivery in the presence of chloroquine varied considerably between different cells, indicating possible different mechanisms of plasmid-peptide internalisation and intracellular trafficking. The efficiency of branched 8Tat peptide as a plasmid DNA carrier varied between different cell types and have to be optimised for each application.