SUPPLEMENTARY METHODS
Plasmid constructs and Adenoviruses. LacZ from pcDNA3.1/His/LacZ (Invitrogem) was
amplified with Platinum pfx DNA polymerase (Invitrogen) using a 5’ primer flanked with
3’
end
of
F2A
fragment
(5’CTTTGAATTTTGACCTTCTCAAGTTGGCCGGAGACGTCGAGTCCA
ACCCCGGGCCTATGGACCCCGTCGTTTTACAAC-3’) and a 3’ primer flanked with
part
of
the
shuttle
vector
pShuttleX
(5’GTTTAAACTTAAGCTTGGTACAATTCTTATTTTTGACAC
CAGACCAACTGGTAA-3’). The PCR product was subcloned into pCR4Blunt-TOPO
vector (Invitrogen) and sequenced at Lone Star Labs Inc. (Houston, TX). The resulting
partial-F2A-LacZ-pshuttlex fragment in pCRBluntII-TOPO was amplified again using a
5’ primer flanked with 3’ end of iMC, previously described (13), an HA-tag, and the
remaining
5’
end
of
the
F2A
(5’CCGGAGTCGACTATCCGTACGACGTACCAGACTACGCACTCGACGTGAAA
CAGACTTTGAATT TTGACCTTCTCAAGTTG-3’) and a 3’ primer flanked with 5’
partial LacZ with a ClaI site (5’-CCACGCTCATCGATAATTTCACCG-3’). This PCR
fragment was cloned into the pCR4Blunt-TOPO vector and sequenced by Lone Star
Labs Inc. This fragment was then excised with BamHI (New England Biolabs, Ipswich,
MA) and ClaI (Invitrogen) and subcloned into similarly digested pCR4Blunt-TOPO
vector containing the partial-F2A-LacZ-pShuttlex fragment described above to make
partialiMC-HA-F2A-LacZ-partialpShuttleX. Next we excised this fragment with SalI
(New England Biolabs) and AflII (New England Biolabs) and subcloned it into the
similarly digested pShuttleX-iMC plasmid, previously described (13), to make
pShuttleX-iMC-HA-F2A-LacZ (also known as bicistronic vector or bv).
pShuttle gene cassettes were subcloned into the Adeno-X expression system (BD
Biosciences) to create E1, E3-deleted, replication-incompetent Ad5 adenoviruses as
previously described (11). Production, purification, and large-scale expansion as well as
titration of adenovirus were carried out by the Vector Development Laboratory Core at
Baylor College of Medicine. Ad5 adenovirus expressing no transgene (Ad-Empty),
EGFP (Ad-EGFP), dsRED (Ad-dsRED) and LacZ (Ad-LacZ) were purchased from the
Vector Development Laboratory Core at Baylor College of Medicine.