**2.1 The pUSF-6 vector**

64 Bacterial Artificial Chromosomes

2008; Volpi, 2005). It is also important to note that the current shingles vaccine has been shown to only reduce the risk of shingles by 50% (Oxman et al., 2005). Because of this, VZV continues to be an important public health concern. In order to improve future prevention and treatment of VZV infections, a better understanding of VZV's biology and pathogenesis

VZV contains the smallest genome among the eight human herpesviruses, consisting of a 125-kb double-stranded DNA genome that encodes 70 unique open reading frames (ORFs). The function of most of these ORFs, however, was largely unknown until recent years. This is in part due to the absence of both a genetic tool to efficiently generate mutant clones for loss-of-function studies and a true animal model for large scale screening of *in vivo* virulence

Obstacles in mutagenizing VZV include its large genome size, narrow host range, and marked differences in replication cycles when studied *in vitro* versus *in vivo* (Arvin, 1996; Cohen, 2001). A once prevalent technique to create recombinant VZV variants was the fourcosmid system, made by cloning overlapping segments of the VZV genome into four large cosmids (Cohen & Seidel, 1993; Mallory et al, 1997; Niizuma et al., 2003). Co-transfection of these cosmids, one of which containing a mutation in the desired ORF, created a recombinant VZV variant. However, this method alone faced many challenges. For example, research was thwarted because co-transfection of the large cosmids into permissive mammalian cells and multiple homologous recombination events within a single cell were

More recent developments have helped to circumvent these problems by cloning the entire VZV genome as a bacterial artificial chromosome (VZVBAC) (Nagaike et al., 2004). This approach provides easy and efficient manipulation of the viral genome and rapid isolation of recombinant viruses, making the systemic deletion of every ORF in the genome feasible. A firefly luciferase cassette is also inserted into the VZVBAC to produce a novel luciferase VZVLuc BAC. This allows us to not only generate VZV ORF deletion mutants, but also

Viral BACs are created when a BAC vector sequence is inserted into the viral genome. Typically, a BAC vector is roughly 10-kb long and contains an origin of replication, genes essential for BAC replication, and genes to control the rate of replication; ideally the copy number should be limited to one or two BACs per bacterial cell (Warden et al., 2010). An antibiotic resistance marker and selection marker, such as a green fluorescent protein, are also added to select for bacterial colonies harboring the BAC vector and isolate these BAC-containing recombinant viruses. BAC vectors in addition must also be flanked by a 500-1000-bp sequence homologous to the target sequence at the site of insertion. Lastly, loxP sites are commonly included at both ends of the BAC sequence to excise the vector after recombinant viruses are generated, as is required for vaccine production (Zhang et

necessary to generate the full-length viral genome (Zhang et al, 2008).

monitor its subsequent growth in cultured cells.

**2. Generation of a VZVBAC**

al., 2007, see below).

is critical.

**1.2 VZV research methods** 

factors (Cohen et al., 2007).

For our purposes, the VZVBAC was constructed from a pUSF-6 vector. As shown in Fig. 1, this vector contains the prokaryotic replication origin (*ori*), replication and partition function (*repE, parA, parB*) genes, chloramphenicol resistance (*camr*) gene, and a green fluorescent protein (*GFP*) gene. Insertion of a GFP reporter gene in BAC DNA is a popular means to visualize *in vitro* infections in cell culture. Viral GFP is expressed using the SV40 early promoter and polyadenylation signals, which activate the gene during the appropriate stages of viral replication and cause the cell to fluoresce (Marchini et al., 2001). The vector is also flanked by two 500-bp VZV fragments and contains a loxP site at each end (Fig. 1).

Fig. 1. The BAC vector, pUSF-6. The vector contains the prokaryotic replication origin *(ori)*, replication and partition (*repE, par,* and *parB*) genes, camr gene, green fluorescent protein (*GFP*) gene, two *loxP* sites and two VZV homologous sequences, a and b. To insert this BAC vector into a VZV cosmid, pUSF-6 was digested by BamHI, resulting in a linear fragment.
