Cloning of replication-incompetent herpes simplex viruses as bacterial artificial chromosomes to facilitate development of vectors for gene delivery into differentiated neurons.

We have previously described the adaptation of a tetracycline-regulated system of gene expression for herpes simplex virus (HSV) vectors and demonstrated that such a system was capable of inducible foreign gene expression in irreversibly differentiated neurons. These studies suggested that such gene delivery vectors would be especially useful for studying the neuron in vitro. Here, we describe the cloning of a replication-incompetent HSV vector as a bacterial artificial chromosome (BAC) to facilitate vector construction. Using prokaryotic genetic techniques for allele replacement, we demonstrate the ease of manipulation of the BAC-containing vector, including the construction of vector mutations for which there is no simple phenotypic selection. Such constructions include the insertion of a tetracycline-regulated gene cassette into the UL41 gene for regulated gene expression and the mutation of the UL48 gene to reduce vector toxicity. In addition, HSV vectors cloned as BACs can be sequentially modified to...