Overall Design Solution

The goal of our project is to engineer an HSV viral genome in the form of a bacterial artificial chromosome (BAC) to produce a customizable viral vector for many purposes. The original BAC that we acquired contains genes that are not ideal for our purpose, including Cre-LoxP recombination sites that are designed for animals or cells expressing Cre enzyme to excise the bacterial artificial chromosome sequence to reconstitute viral genome, as well as a beta-galactosidase gene used for selection marker in bacterial and mammalian cells. We would like to use the viral vector potentially with other viral vectors that utilize Cre-LoxP system, therefore an orthogonal system is preferred. In this case, the FRT-flp expression system was chosen. The FRT-flp system functions similarly to Cre-LoxP: the expression of the flp enzyme would delete the sequence flanked by two FRT sites. On the other hand, we would like to use the beta-gal gene as a selection marker for a different part of the genome; therefore, the gene needs to be knocked out. Secondly, the virus is inherently toxic to mammalian cells. The uncontrolled spread of the virus would be fatal. To control the toxicity, the thymidine kinase gene, a gene that is crucial to viral replication, will be replaced with a Tn7 DNA insertion site. The site would allow for the insertion of any DNA construct into the viral genome. The final design should be a new bacterial chromosome that can be customized as a gene delivery tool.

Modification 1: Knockout Blue Gene

Modification 2: Knockout tk Gene with Insertion Site