**4.2 Generation of a VZV deletion clone**

70 Bacterial Artificial Chromosomes

Fig. 5. Monitoring VZVLuc virus replication in SCID-hu mice. (A) SCID-hu model. 1. Human fetal thymus/liver tissues were implanted under SCID mouse kidney capsule. 2. Two to three months later, the implant was inoculated with VZVLuc. 3. Viral replication in human T

thymus/liver implants in SCID mice. Three SCID-hu mice with thymus/liver implants were inoculated with VZVLuc. Using IVIS, each mouse was scanned daily (from day 0 to day 8). Measurements were taken 10 minutes after i.p. injection with luciferin substrate. Only images from one mouse are shown. Warmer colors indicate higher viral load; colder colors indicate lower viral load. (C) VZV growth curves *in vivo*. Bioluminescence from three SCIDhu mice in the above experiment (B) was measured and VZV growth curves in human

cells was detected by IVIS. (B) Replication and progression of VZVLuc in human

**4. Generation of recombinant VZV using a highly efficient homologous** 

To test the novel VZVLuc system for studying VZV pathogenesis, five single ORF deletion mutants were first generated, starting from ORF0 to ORF5, via the homologous recombination system harbored in DY380 *E. coli*. Afterwards, the VZVLuc was used for genome-wide mutagenesis to systematically delete each individual VZV ORF for functional

The DY380 *E. coli* strain offers a highly efficient homologous recombination system for chromosome engineering by enabling efficient recombination of homologous sequences as short as 40-bp (Yu et al., 2000). A defective lambda prophage supplies the function that protects and recombines linear DNA. In addition, the system is strictly regulated by a temperature-sensitive lambda repressor. This allows homologous recombination between two sequences to be transiently induced by activating the prophage through incubation at

thymus/liver implants were generated.

characterization of the VZV genome.

**4.1 The DY380** *E. coli* **strain** 

42°C for 15 minutes.

**recombinant system** 

The entire process to engineer a VZV ORF deletion mutant (ORFXD) is illustrated in Fig. 6. VZVLuc BAC DNA was first introduced into DY380 by electroporation. Homologous recombination functions were transiently induced by increasing the culturing temperature to 42°C for 15 minutes during electroporation-competent cell preparation. A kanR expression cassette was amplified from pGEM-oriV/kanR by PCR using two primers containing 40-bp homologous sequences flanking the target ORF (ORFX). The PCR product was then transformed into the DY380 harboring the VZVLuc BAC via electroporation. As expected, homologous recombination occured between the ORF flanking sequences of the cassette and targeted ORF, replacing the ORFX with the kanR gene and generating an ORFXD VZV clone (Zhang et al., 2008).

Fig. 6. Generation of a VZV deletion clone. (A) The DY380 strain permits transient induction of recombination system by incubation at 42°C for 15 min during electro-competent cell preparation. VZVLuc BAC DNA was introduced into DY380 by electroporation. (B) Amplification of the kanR expression cassette by PCR using a primer pair to add 40-bp homologous sequences flanking ORFX. (C) 200ng of the above PCR product was transformed into DY380 carrying the VZVLuc BAC by electroporation. (D) Homologous recombination between upstream and downstream homologies of ORFX replaced ORFX with the kanR cassette, creating the ORFX deletion VZV clone. (E) Recombinants were selected on LB agar plates. (F) The deletion of ORFX DNA was isolated and confirmed by testing antibiotic sensitivity and PCR analysis. The integrity of the viral genome after homologous recombination was examined by restriction enzyme digestion. (G) Purified BAC DNA was transfected into MeWo cells. (H) 3-5 days after transfection, the infected cells were visualized by fluorescence microscopy.

Successful ORF deletion clones were confirmed by three sequential procedures: 1. antibiotic sensitivity selection, 2. mini-preparation of BAC DNA with PCR verification, and 3. maxipreparation of BAC DNA with HindIII digestion profiling. Firstly, recombinants were selected on LB plates with chloramphenicol or kanamycin for resistant colonies. It was also important to verify that the deletion clones were sensitive to ampicillin since ampicillin-resistant circular pGEM-oriV/kanR was used as the PCR template. Multiple colonies were then selected for mini-preparation of BAC DNA to confirm the ORF deletion and kanR replacement by PCR. Lastly the PCR-verified clones were chosen for maxi-preparation of BAC DNA and digested with HindIII to ensure that only the targeted sequence was deleted. When the digestion pattern of the deletion clone was compared to that of the parental WT VZVLuc clone, no additional deletions from the genome were detectable (as shown in Fig. 3G).

Finally, to generate VZV deletion mutant viruses, these verified clones were transfected into MeWo cells, along with WT VZVLuc DNA. The size and growth kinetics of the virus as measured by resultant plaques, or absence of plaques, are indicative of the essentiality of a particular VZV ORF for viral replication, discussed later.
