Fig. 1. Restriction endonuclease (HindIII) analysis of eight transposed bacterial artificial chromosomes containing the Bovine herpesvirus 1 genome. HindIII digestion fragments were resolved in 0.7% agarose gel for 20.5 h at 2 V/cm. Lanes 1-8, pBAC-BHV37::Tn(EGFP-KanR)-1-8. Molecular weight marker, 1-kb DNA ladder (Invitrogen).

6. The monolayers were rinsed once with approx 0.5 mL/well of Opti-MEM and as much of the wash solution as possible was removed.

7. Each reaction was diluted to 1 mL using Opti-MEM, and the mixture was added to the washed monolayer.

8. The transfection reactions were incubated for 18-24 h at 37°C with 5% C02.

9. The transfection reaction was gently aspirated from the monolayer and replaced with 3 mL of growth media supplemented with 10% donor calf sera and 2 mM NN-HBA (9) and incubated at 37°C with 5% C02.

10. The transfections were monitored for GFP expression, generally visible after 48 h, and for cytopathic effects (CPEs) typical of BoHV-1 infection, usually observed 3-7 d posttransfection (see Note 8).

11. If no CPE was evident at 7 d post transfection, the supernatant was passaged three times to confirm the noninfectious virus phenotype.

12. Cell supernatants were passaged by preparing a fresh CRIB-1 monolayer as described for the transfection experiments.

13. Following removal of the growth medium (Subheading 3.3.2., step 1), the monolayers were washed once with PBS. After washing, a 200-|L aliquot of the transfection supernatant was added to the monolayer followed by incubation at 37°C for 1 h, with gentle rocking every 15 min.

14. After 1 h the liquid was removed, replaced with 2 mL of growth medium, and subsequently incubated at 37°C with 5% C02 for 7 d.

15. This procedure was repeated a further two times unless CPEs became evident at an earlier passage.

3.3.3. Sequence Analysis of Transposition Site

1. Sequencing of the transposed BAC requires highly purified DNA. BAC DNA for automated sequence analysis is prepared from a single colony inoculated into 250 mL of LB-Cap/Kan followed by incubation at 37°C overnight with shaking.

2. BAC DNA was recovered using the NucleoBond BAC 100 kit (Macherey-Nagel) according to the manufacturer's instructions. At least 1 |g DNA is used per sequencing reaction.

3. BAC DNA was prepared for sequencing by heating 1 | g DNA at 60°C for 30 min (see Note 9).

4. To the heat-treated BAC DNA, 16 |L Big-Dye terminator V2 mix, 2 |L DMS0, 20 |M KanME, 5'-CTCCTTCATTACAGAAACGGC-3' were added, and the volume was adjusted to 40 ||L using sterile MQW (see Note 10).

5. The sequencing cycling conditions used were: (95°C, 5 min) for 1 cycle; (95°C, 30 s; 50°C, 20 s; 60°C, 4 min) for 60 cycles; (60°C, 4 min) for 1 cycle; then held at 4°C.

6. Following completion of cycling, the entire sequencing reaction was added to a microfuge tube containing 100 |L absolute ethanol and 4 |L 3 M sodium acetate, pH 4.6, and incubated at RT for 5 min.

7. Sequencing products were pelleted at 10,000g for 30 min at RT.

8. The supernatant was aspirated, and the pellet was gently washed with 75% ethanol.

Table 3

Characterization of the Sites of Transposition in Eight Modified Bovine herpesvirus 1 Infectious Clones

GFP Gene

Clone0 Infectious6 expression Insertion site functionc

Table 3

Characterization of the Sites of Transposition in Eight Modified Bovine herpesvirus 1 Infectious Clones




UL43 (virion protein)


0 0

Post a comment