The transfer of foreign genes into eukaryotic cells, in particular mammalian cells, has been essential to our understanding of the functional significance of genes and regulatory sequences as well as the development of gene therapy strategies. To this end, different mammalian expression vector systems have been designed. The choice of a particular expression system depends on the nature and purpose of the study and will involve selecting particular parameters of expression systems such as the type of promoter/enhancer sequences, the type of expression (transient versus stable) and the level of desired expression. In addition, the success of the study depends on efficient gene transfer. The purification of the expression vectors, as well as the transfer method, affects transfection efficiency. Numerous approaches have been developed to facilitate the transfer of genes into cells via physical, chemical or viral strategies. While these systems have all been effective in vitro they need to be optimized for individual cell types and, in particular, for in vivo transfection.

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A 511 (+/-) charge ratio was found to be the optimal ratio for transfection of both ss-and dsDNA. After a 5 h exposure, 7.51 +/- 0.89% of the radioactivity was associated with the nuclear fraction whereas only 1.07 +/- 0.23%, was found in the nuclear fraction when dsDNA was used. The nuclear radioactivity detected after a 24 h exposure was only 1/3 of that after 5 h. Analysis of fragment stability in the cytosolic and nuclear fractions showed the presence of intact fragment in each subcellular compartment. No intranuclear/intracellular fragment could be detected in control experiments with naked DNA. Conclusions. The results from these experiments indicate that small fragments of DNA can be efficiently and rapidly transferred intact to the cell nucleus using cationic lipids and that ssDNA fragments are more effective than dsDNA fragments for nuclear delivery.

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There are several advantages, both in vitro and in vivo , in utilizing bacteria that express a fluorescent protein. Such a protein can be transiently incorporated into the bacteria or integrated within the bacterial genome. The most widely utilized fluorescent protein is green fluorescent protein (GFP), but limitations exist on its use. Additional fluorescent proteins have been designed that have many advantages over GFP and technologies for their incorporation into bacteria have been optimized. In the current study, we report the successful integration and expression of a stable fluorescent reporter, mCherry (red fluorescent protein, RFP), into the genome of a human pathogen, Group A Streptococcus pyogenes (GAS) isolate AP53(S-). RFP was targeted at the atg codon of the fcR pseudogene that is present in the mga regulon of AP53(S-). Transcription of critical bacterial genes was not functionally altered by the genomic integration of mCherry. Host virulence both in vitro (keratinocyte infection and cytotoxicity) and in vivo (skin infection) was maintained in AP53(S-)-RFP. Additionally, survival of mice infected with either AP53(S-) or AP53(S-)-RFP was similar, demonstrating that overall pathogenicity of the AP53(S-) strain was not altered by the expression of mCherry. These studies demonstrate the feasibility of integrating a fluorescent reporter into the bacterial genome of a naturally virulent isolate of Group A S. pyogenes for comparative experimental studies.