The cell-specific expression of herpes simplex virus 1 thymidine kinase (HSV-1-tk) has provided a simple and highly efficient technique to achieve conditional ablation of targeted cell types in transgenic mice. The ablation is induced by treating transgenic animals expressing HSV-1-tk with the antiherpetic drug ganciclovir. In lymphoid tissues of mice expressing HSV-1-tk from an immunoglobulin promoter, administration of ganciclovir leads to massive destruction of Band T-cell lineages. Tissues not expressing HSV-1-tk are insensitive to drug treatment. After depletion of >99% of total thymocytes, a number of progenitor cells remain that are able to repopulate all T-cell lineages within 7 days. The ability to control and direct ablation allows for creation of conditional mutant phenotypes at precise periods of development. This technique also provides a potential means to enrich stem cell populations as well as permitting the creation of animal models for particular pathological conditions. An understanding of the self-renewing nature of stem cells and the events that trigger their differentiation represents a major challenge to developmental biology. Our laboratory and others have exploited the creation of transgenic animals as a strategy to study vertebrate development. Utilizing tissue-specific promoters and enhancers, it is possible to direct expression of cloned genes to restricted cell types enabling the experimental manipulation of cellular and organ physiology (1,2). A reciprocal to this augmentation approach would be the creation of a transgenic hypomorph (i.e., a mouse deficient in a particular cellular function). This would allow an alternative means to evaluate the contribution of a cell or gene to a particular developmental program. To address this problem, we have developed a method for creating transgenic hypomorphs that results in the conditional ablation of specific cell types during defined periods of development and differentiation. Our strategy involves expression of an enzyme that is not itself deleterious but is capable of metabolizing a benign substrate to a toxic product.One potential candidate for this strategy is the herpes simplex virus 1 thymidine kinase (HSV-1-tk). HSV-1-tk is by itself not harmful to mammalian cells. However, in contrast to mammalian thymidine kinase, HSV-1-tk is capable of phosphorylating specific nucleoside analogs, such as acyclovir (ACV), to the nucleoside monophosphate (3, 4). The nucleoside monophosphate is phosphorylated by cellular kinases to the nucleoside triphosphate and incorporated into DNA leading to inhibition of DNA synthesis and cell death (4,5). The high selectivity of the viral thymidine kinase for these nucleoside analogs and the very low affinity of mammalian thymidine kinase for these nucleosides make it possible to selectively kill HSV-1-tk-expressing cells among a population of dividing cells.We have demonstrated (6) the feasibility of this conditional-ablation approach in cell lines transfected with the gene for HSV-1-tk. The success o...

A cancer treatment is described in which i.m. injection of plasmid DNA (pDNA) encoding murine interferon ␣ (mIFN-␣) leads to potent antitumor effects on primary and metastatic tumors in mice. Mice bearing s.c. B16F10 melanoma, Cloudman melanoma, or glioma 261 tumors were injected i.m. with mIFN-␣ pDNA. In all three tumor models, a significant reduction in tumor volume and enhancement of survival was found after IFN pDNA therapy. The mIFN-␣ pDNA could be injected as infrequently as once every other week and still produce a significant antitumor effect, and, in a metastatic tumor model, the therapy markedly reduced the number of lung tumor metastases. Depletion of immune cell subsets indicated that CD8 ؉ T cells were required for the antitumor response. These studies demonstrate that primary and metastatic tumors can be treated systemically by i.m. injection of a plasmid encoding a cytokine gene.

Inteins are polypeptide sequences found in a small set of primarily bacterial proteins that promote the splicing of flanking pre-protein sequences to generate mature protein products. Inteins can be engineered in a "split and inverted" configuration such that the protein splicing product is a cyclic polypeptide consisting of the sequence linking two intein subdomains. We have engineered a split intein into a retroviral expression system to enable the intracellular delivery of a library of random cyclic peptides in human cells. Cyclization of peptides could be detected in cell lysates using mass spectrometry. A functional genetic screen to identify 5-amino acid-long cyclic peptides that block interleukin-4 mediated IgE class switching in B cells yielded 13 peptides that selectively inhibited germ line ⑀ transcription. These results demonstrate the generation of cyclic peptide libraries in human cells and the power of functional screening to rapidly identify biologically active peptides.

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