Lentiviral vectors with amplified beta cell-specific gene expression.

An important goal of gene therapy is to be able to deliver genes, so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Although tissue-specific promoters confer selectivity, in a vector-based system, their activity may be too weak to mediate detectable levels in gene-expression studies. We have used a two-step transcriptional amplification system to amplify gene expression from lentiviral vectors using the human insulin promoter. In this system, the human insulin promoter drives expression of a potent synthetic transcription activator (the yeast GAL4 DNA-binding domain fused to the activation domain of the Herpes simplex virus-1 VP16 activator), which in turn activates a GAL4-responsive promoter, driving the enhanced green fluorescent protein reporter gene. Vectors carrying the human insulin promoter did not express in non-beta-cell lines, but expressed in murine insulinoma cell lines, indicating that the human insulin promoter was capable of conferring cell specificity of expression. The insulin-amplifiable vector was able to amplify gene expression five to nine times over a standard insulin-promoter vector. In primary human islets, gene expression from the insulin-promoted vectors was coincident with insulin staining. These vectors will be useful in gene-expression studies that require a detectable signal and tissue specificity.

Phage R4 integrase mediates site-specific integration in human cells.

The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.

Toxicity of the bacterial luciferase substrate, n-decyl aldehyde, to Saccharomyces cerevisiae and Caenorhabditis elegans.

This study determined that the bacterial luciferase fusion gene (luxAB) was not a suitable in vivo gene reporter in the model eukaryotic organisms Saccharomyces cerevisiae and Caenorhabditis elegans. LuxAB expressing S. cerevisiae strains displayed distinctive rapid decays in luminescence upon addition of the bacterial luciferase substrate, n-decyl aldehyde, suggesting a toxic response. Growth studies and toxicity bioassays have subsequently confirmed, that the aldehyde substrate was toxic to both organisms at concentrations well tolerated by Escherichia coli. As the addition of aldehyde is an integral part of the bacterial luciferase activity assay, our results do not support the use of lux reporter genes for in vivo analyses in these model eukaryotic organisms.

Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase.

We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.

Design and application of a biosensor for monitoring toxicity of compounds to eukaryotes.

Here we describe an alternative approach to currently used cytotoxicity analyses through applying eukaryotic microbial biosensors. The yeast Saccharomyces cerevisiae was genetically modified to express firefly luciferase, generating a bioluminescent yeast strain. The presence of any toxic chemical that interfered with the cells' metabolism resulted in a quantitative decrease in bioluminescence. In this study, it was demonstrated that the luminescent yeast strain senses chemicals known to be toxic to eukaryotes in samples assessed as nontoxic by prokaryotic biosensors. As the cell wall and adaptive mechanisms of S. cerevisiae cells enhance stability and protect from extremes of pH, solvent exposure, and osmotic shock, these inherent properties were exploited to generate a biosensor that should detect a wide range of both organic and inorganic toxins under extreme conditions.