Anderton at al Supplementary Figure Legends:
Supplementary Figure 1 Schematics showing how individual EBNA3 genes
were deleted from the EBV-BAC. (A) Sections of the EBV genome were
amplified by PCR. The coordinates of PCR products (based on the B95.8 strain
of EBV, Acc#:V01555) and their approximate exon locations are shown. Pairs of
these products were cloned contiguously to generate shuttle plasmids containing
a fragment of the EBV genome with an internal deletion of EBNA3A (3AKO),
EBNA3B (3BKO) or EBNA3C (3CKO). The location of Eco RI, Cla I and Not I
restriction sites within this region are also indicated. (B) Deletion of each EBNA3
gene requires two rounds of recombination. First, recombination occurs between
one PCR product within the shuttle plasmid (3AKO is shown here as an example)
and either of the homologous regions in the EBV-BAC. If this first recombination
occurs at the homologous region at the Start of the gene, the cointegrants are ‘S’
oriented (I). If the first recombination occurs at the homologous region at the End
of the gene, the cointegrants are ‘E’ oriented (II). Either cointegrant can then
undergo a second recombination. If this occurs at the same site as the original
recombination event (A), the cointegrant will revert back to the original EBV-BAC
and the original shuttle plasmid. If the second recombination event occurs
between the PCR product not used for the first recombination and its
corresponding homologous sequence in the EBV genome, then the result will be
the desired recombinant EBV-BAC (B). In this example, this leads to the deletion
of EBNA3A.
Supplementary Figure 2 Screening of EBNA3-knockout (KO) and revertant
(Rev) BACs. BAC DNA was isolated by miniprep and analysed by restriction
digest with Cla I or Not I followed by pulsed field gel electrophoresis. (A) Deletion
of individual EBNA3 genes. The original EBV-BAC along with cointegrants of
both ‘E’ and ‘S’ orientations are shown. Deletion of EBNA3A introduces an
additional Cla I restriction site meaning two fragments of 24.5 and 38.5 kb (lane
5) are produced instead of the 66 kb fragment present in the EBV-BAC (lane 2).
Deletion of EBNA3B introduces an additional Not I site giving rise fragments of
15 and 19 kb (lane 10) instead of 37 kb (lane 7). Deletion of EBNA3C introduces
an additional Cla I site giving rise to fragments of 31 and 32 kb (lane 16 and 17)
instead of 66 kb (lane 12). 3CKO+BamW (which arose from cointegrant ‘S’, lane
13) has approximately two more copies of the Bam W repeat than 3CKO (which
arose from cointegrant ‘S’, lane 14) or the original EBV-BAC. (B) Generating
revertants from knockout-BACs. Restriction digest shows revertants to be
identical to the original EBV-BAC. Comparing KOs and Revs, a 36 kb fragment is
restored in 3ARev and a 66 kb fragment in 3BRev and 3CRev.
Supplementary Figure 3 Screening of BACs recovered from 293 producer cell
lines. Episomal DNA was isolated and analysed by restriction digest with Eco RI
followed by pulsed field gel electrophoresis. The BAC DNA recovered from a
selection of 293 cell clones is shown to be identical to the BAC originally
transfected (i.e. 293-3AKO is identical to 3AKO-BAC). In the case of 293 cells
harbouring revertant BACs, recovered DNA was compared to the EBV-BAC
rather than the revertant BACs originally transfected. Deletion of EBNA3B or
EBNA3C from the EBV-BAC caused a decrease in the size of the 30 kb Eco RI
fragment to either 27 or 28 kb respectively. Deletion of EBNA3A from the EBVBAC resulted in changes to the Eco RI restriction fragment pattern that cannot be
detected by this technique (i.e. the 3AKO-BAC and EBV-BAC appear identical in
this figure).