![]() M., Fragkos, M., Palaszewski, I., and Coutelle, C. (2002) CpG-depleted plasmid DNA vectors with enhanced safety and long-term gene expression in vivo. (2004) Nonmethylated CG motifs packaged into virus-like particles induce protective cytotoxic T cell responses in the absence of systemic side effects. Storni, T., Ruedl, C., Schwarz, K., Schwendener, R. (2005) Potential oncogene activity of the woodchuck hepatitis post-transcriptional regulatory element (WPRE). (1983) Purification of genomic sequences from bacteriophage libraries by recombination and selection in vivo. (1995) A DNA segment conferring stable maintenance on R6K gamma-origin core replicons. Wu, F., Levchenko, I., and Filutowicz, M. (2007) pPSX: a novel vector for the cloning and heterologous expression of antitumor antibiotic gene clusters. (1986) Broad host range vectors derived from an RSF1010::Tn1 plasmid. (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. (1977) Construction and characterization of new cloning vehicles. ![]() Ampicillin-resistant derivatives of the plasmid pMB9. restriction endonuclease digestion of ligation mixtureīolivar, F., Rodriguez, R.modulation of immune response in cancer gene therapy.In this chapter, the emphasis is placed on efficient and flexible versions of DNA cloning protocols using selection of recombinant plasmids by restriction endonucleases directly in the ligation mixture. Rapid selection of a desired recombinant plasmid against a background of other plasmids continues to be a challenge. Techniques relying on site-specific or homologous recombination are preferred for construction of large plasmids (>15 kb), while digestion of DNA by restriction enzymes with subsequent ligation of the resulting DNA fragments continues to be the mainstream approach for generation of small- and medium-size plasmids. Optimal immune response to the plasmid vectors can be modulated via inclusion or exclusion of DNA sequences containing immunostimulatory CpG sequence motifs.ĭNA fragments facilitating construction of plasmid vectors should also be considered for inclusion in the design of plasmid vectors. The use of appropriate promoters, other regulatory elements, and mammalian maintenance devices ensures that the therapeutic gene or genes are adequately expressed in target human cells. Structural and maintenance plasmid stability in bacteria is required for the plasmid DNA production and can be achieved by carefully choosing a combination of the therapeutic DNA sequences, replication origin, selection marker, and bacterial strain. The plasmids are propagated in bacteria, so, in addition to their therapeutic cargo, they necessarily contain a bacterial replication origin and a selection marker, usually a gene conferring antibiotic resistance. Nonviral gene therapy vectors are commonly based on recombinant bacterial plasmids or their derivatives.
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