Biology Reference
In-Depth Information
Figure 1. Schematic of construct for recombination into deletion III locus of MVA, based on pBELO-
BAC11.
DIIIL and DIIIR = left and right flanks for homologous recombination into MVA genome; GFP =
green fluorescent protein gene; p4B = Fowlpox virus late promoter; CmR = chloramphenicol acetyltransferase
gene for selection in E. coli. Flanking FseI sites are absent from MVA and permit excision of the cassette. The
PacI site is provided for linearisation of the plasmid prior to recombination with MVA in infected transfected
cells.
Generation of MVA-BAC
CEF cells in 6-well plates were infected with MVA-BAC-parent at 5 pfu/cell and
2 h later the inoculum was replaced with growth medium containing 45 µM
isatin-
β
-thiosemicarbazone (IβT).
β
T). The compound was kindly synthesised by Dr
J. Robertson (Chemistry Research Laboratory, Oxford University) and was dis-
solved to 5 mg/mL in acetone for storage at −20°C, then diluted to 1mg/mL in
0.25 M NaOH immediately prior to use. The cells in some wells were transfected
with pCI-Cre (obtained from A. Domi, NIAID, NIH, Bethesda, MD) using Li-
pofectamine (Invitrogen). The DNA was phenol-extracted from the cultures 24h
later as described [53] and resuspended in 20 µl of Tris-EDTA. Of this material,
3 µl was electroporated into DH10B E. coli (Invitrogen) prior to selection on LB
plates with chloramphenicol (12.5 µg/mL) and miniprep by alkaline lysis from
liquid LB cultures. Clones were screened by PCR for the presence of the genes
MVA005, MVA010, MVA044, MVA086 and MVA188. For restriction mapping
and pulsed-field gel electrophoresis, BAC DNA was prepped by QIAgen kit and
0.5-1 µg of DNA was digested overnight in a 15 µl reaction with the relevant en-
zymes (NEB). MVA-BAC clone 26 was shotgun sequenced at eightfold coverage
by Lark Technologies (Cogenics), Inc.
bAc rescue
BHK-21 cells (maintained in DMEM+10% FCS) were seeded into 6-well plates
and infected with FP9-LacZ [58], an attenuated strain of Fowlpox virus expressing
β
-galactosidase, at 1 pfu/cell in Optimem (Gibco). After 1-2 hours, the cells were
transfected with 4 µg of Qiagen-purified MVA-BAC DNA using Lipofectamine
2000 (Invitrogen) in Optimem. The BAC DNA was stored at 4°C, was never
vortexed, and was pipetted only with cut-off tips. The cells were monitored for
GFP expression by epifluorescence microscopy and were harvested by freeze-thaw
4-6 days later. The lysate was used to infect fresh BHK cell monolayers and the
cultures were monitored for the appearance of virus growth. Rescued viruses were
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