Human Herpes Virus 8 (HHV-8) Mutagenesis.

Description of projects available to graduate students:
Project 1. Construction of mutated HHV-8 thymidine kinase gene containing a functional green fluorescent protein gene. As part of our interest in generating HHV-8 site-specific mutants, we need to generate a recombinant plasmid that contains the HHV-8 gene for thymidine kinase (TK) and its natural promoter. This will be accomplished by isolation of the tK gene and its promoter sequence through either direct PCR amplification or restriction enzyme digestion of viral DNA. The isolated TK gene will be cloned into pUC19 and functional TK activity determined by transient expression assay of human 143 TK- cells. Once the TK clone has been demonstrated to express a functional TK gene, we will insert the GFP gene under the control of the CMV IE promoter into the TK coding sequences. Expression of GFP will be determined by transient expression in Vero cells. Once constructed, the GFP-mutated HHV-8 TK gene will be used in future experiments designed to construct a TK- HHV-8 virus.

Techniques graduate student will learn:
Cloning, DNA isolation, restriction enzyme digestion, southern blotting, transfection of mammalian cells.