Download Supplemental figure 1 Complete CLSM stacks of Ad3 texas

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Supplemental information Materials and Methods (Construction of the Ad∆EPTETP vectors).
recombination
The new Ad∆EP-TETP vectors were generated by homologous
between
the
Eco RV-linearised
adenoviral
transfer
plasmids
ppolyAd∆EP-TETP, ppoly-Ad∆EP-TETP-∆24, ppolyAd∆EP-TETP-∆19, ppoly-Ad∆EPTETP-∆24∆19, and a 28 kb Cla I- Pac I fragment derived from the ppoly-H5dl324,
following the procedure of Chartier et al1. Mutations in the E1 were first introduced
into the starting transfer plasmid ppolyAd∆EP-TETP.
This low copy plasmid2
contains the left ITR sequence followed by an artificial packaging signal, four copies
of the mouse tyrosinase enhancer element (TE) fused to the human tyrosinase
promoter (TP) replacing the endogenous E1A promoter (EP)3. Further, this plasmid
contains the E1/pIX region up to nucleotide 4 161 (wt Ad5) separated by a unique
Eco RV site from 800 bp of the Ad5 right end, including the right ITR. The E1B19 kDa gene deletion encompassing 146 bp (dl 337) was introduced by a Sac IBstE II-deletion into the E1B gene4.
In order to introduce this deletion into
ppolyAd∆EP-TETP transfer plasmid, a 709 bp Xba I- Kpn I-fragment encompassing
nucleotides 1 346 to 2 053 of the Ad5E1 region was subcloned into a Sac I-deleted
pBluescript (pBLSK, Invitrogen) to give rise to pBLSK∆Sac I-XK-frg.
The Sac I-
BstE II-digestion of the resulting plasmid was followed by blunting and re-ligation,
resulting in pBLSK∆Sac I-XK-∆E1B. The sequence deletion was confirmed using
sequencing primer #367 (Supplemental Table 1). The 560 bp Xba I- Kpn I-fragment
containing this deletion was used to replace the according sequence in ppolyAd∆EPTETP, giving rise to ppolyAd∆EP-TETP-∆19. The E1A 122-129 deletion of 24 bp
(∆24)5 was introduced by site-directed mutagenesis.
Deletion containing primers
357 and 358 and restriction-site containing primers 374 (Bst BI-site) and 356
(Acc I-site) (Supplemental Table 1) were used to generate in a 2-step PCR reaction a
746 bp fragment which then was used to replace the parental sequence of
ppolyAd∆EP-TETP and ppolyAd∆EP-TETP-∆19, resulting in ppoly-Ad∆EP-TETP-∆24
and ppoly-Ad∆EP-TETP-∆24∆19, respectively.
The 24 bp deletion and all other
modifications were confirmed by sequencing using the primers indicated in
Supplemental Table 1.
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The 28 kb Cla I- Pac I fragment derived from ppoly-H5dl324 fragment and used for
homologous recombination between the Eco RV-linearised form of the different
ppolyAd∆EP-TETP vectors contained Ad5 sequence starting from 3 333 to the right
end (35 935), except the E3 region, which is deleted in this construct.
For
homologous recombination, 500 ng of the linearised plasmid and 100 ng of the 28 kb
fragment were transformed using freshly prepared calcium-competent E. coli BJ5183
recBC sbcBC1. Correct forms of the resulting ppoly-Ad∆EP-TETP∆19, ppoly-Ad∆EPTETP-∆24 and ppoly-Ad∆EP-TETP-∆24∆19 plasmids were amplified in E. coli
MC1061. To generate infectious virus, 20 g of the ppoly plasmids were digested
with Pac I to release viral DNA genome, followed by transfection of helper 911 helper
cells by the calcium phosphate method.
The DNA precipitate was incubated
overnight, and cells were overlaid with agarose / medium. Plaques appeared within
ten days and were amplified on 911 cells. Viruses were grown to large scale, purified
by two subsequent CsCl-gradient centrifugations, dialysed against PBS and plaquetitered on 911 cells, as described6. Viral titers are summarized in Table 2.
The ppoly plasmids encompassing the sequences for Ad∆EP-TETP-RGD, Ad∆EPTETP-∆24-RGD,
and
Ad∆EP-TETP-∆24∆19-RGD
were
generated
by
two
subsequent homologous recombination steps. The first recombination was between
the Eco RV-linearised form of the three plasmids ppolyAd∆EP-TETP, ppoly-Ad∆EPTETP-∆24, ppoly-Ad∆EP-TETP-∆24∆19 and a modified form of the 28 kb Cla I- Pac I
fragment containing a Swa I restriction site inserted into the deleted fiber region 7.
The second recombination was between the Swa I-linearised form of the above
intermediate plasmids and a fragment EcofrgAd5fiberRGD consisting of 6 756 bp of
the Ad5-right end (E3 deleted) including the ITR sequence plus 8 bp of ppoly
backbone sequence. In a preceding step, the RGD-modified fiber sequence was
introduced into the 6 729 bp EcofrgAd5fiber using overlapping PCR and an exchange
of the fragment flanked by the unique Mfe I and Nhe I sites (primers #189, #190,
#430, #759, Supplemental Table 1)7.
Digestion with Pac I to release the viral
genome and subsequent generation of viruses were performed as described above.
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Several attempts to construct a fiber chimeric Ad∆EP-TETP-∆24∆19-F35 by an
analogous procedure as described above for the RGD fiber-modified viruses failed,
which is why we decided to include the E3 region in this construct, as Ad5F35 fiberchimeric vectors containing the complete E3 region had been constructed with
success previously8.
For the generation of the fiber containing fragment, the
6 729 bp EcofrgAd5fiber was first extended to a 9 442 bp SpeEcofrgAd5F35
fragment containing the E3 region (starting at wt Ad5 sequence 27 082). Second, the
sequence encoding the chimeric fiber consisting of 46 amino acids of the Ad5 tail
fused to 278 amino acids of the Ad35 shaft and knob was generated using
overlapping PCR (primers #430, #431, #834 to #837, Supplemental Table 1)7,
followed by an exchange of the fragment flanked by the unique Nde I and Afl II sites.
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Supplemental Table 1. Primers used for plasmid construction and sequencing
Nr
#150
#189
Sequence
tctctagacacaggtgat
cttgggcagaaacagtctccgcggcagtc
acaagttgtgtctcctgtttcctg
acaacttgtgactgccgcggagactgtttct
gcccaagtgcatactctatgtc
catgtttgagagaaaaatgg
cgtggagactggatcctcg
gatttgaccttgcaaatgag
cataatttttcacttactgtagac
Length
18
53
43
#364
#367
#368
#369
#370
#371
#372
#373
#374
cttgtaccggaggtgatcgatccacccagt
gacgacgaggatg
catcctcgtcgtcactgggtggatcgatca
cctccggtacaag
gcaggagcagagccc
tagactctcatttgcaagg
ccacctacccttcacg
ggtgggtttggtgtgg
gttaaatggggcggggc
ctgggaacggggcc
ccattcacgtagccagc
catcaggttgattcatcgg
ccactggtgggatacgagcc
#430
catgaacttaagtgagctgcc
21
#431
catccgcacccactatcttc
20
#759
cattgccacccaaggacc
18
1
Chartier C, Degryse E, Gantzer M, Dieterle A, Pavirani A, Mehtali M. Efficient
generation of recombinant adenovirus vectors by homologous recombination
in Escherichia coli. J. Virol. 1996; 70: 4805-4810.
2
Lathe R, Vilotte JL, Clark AJ. Plasmid and bacteriophage vectors for excision
of intact inserts. Gene 1987; 57: 193-201.
#190
#316
#351
#352
#356
#357
#358
53
20
19
20
24
43
15
19
16
16
17
14
17
19
20
Purpose
SP Ad5, bp 1,329 rev.
SDM rev primer for introduction of RGD4C motif into HI loop.
SDM fwd primer for introduction of RGD4C motif into HI loop.
SP Ad5, bp 35,075 rev.
SP TETP, bp 232 fwd in ppoly.
SP TETP, bp 1,042 rev in ppoly.
SP Ad5, bp 1,106 rev, also used for
SDM.
SDM fwd primer introducing ∆24 E1A
deletion.
SDM rev primer introducing ∆24 E1A
deletion.
SP Ad5, bp 2,207, fwd.
SP TETP, bp 1,032, fwd in ppoly.
SP Ad5, bp 692, fwd.
SP Ad5, bp 1,162, fwd.
SP Ad5, bp 1,663, fwd.
SP Ad5, bp 2,633, fwd.
SP Ad5, bp 3,142, fwd.
SP Ad5, bp 35,326, fwd.
SP TETP, bp 1,195, fwd in ppoly, also
used for SDM.
SDM rev primer, containing Afl II site,
priming site in wt Ad5 at bp 33'095.
SDM fwd primer containing Nde I site,
priming site in Ad5 wt at bp 31'029.
Fwd SP for fiber gene region, priming site
in wt Ad5 at bp 31'487.
#834
gagagtccccctggggtacttactttaaaat 41
SDM fwd primer, introducing left 5/F35
gtttaacccc
crossing in Ad5 wt at bp 31'179.
#835
ggggttaaacattttaaagtaagtacccca 41
SDM rev primer, introducing left 5/F35
gggggactctc
crossing in Ad5 wt at bp 31'162.
#836
cattacagaagacgacaactaaagaatc 44
SDM fwd primer, introducing right 5/F35
gtttgtgttatgtttc
crossing in Ad5 wt at bp 32'809.
#837
gaaacataacacaaacgattctttagttgt 44
SDM rev primer, introducing right 5/F35
cgtcttctgtaatg
crossing in Ad5 wt at bp 32'789.
SP: Sequencing primer; SDM: site-directed mutagenesis; fwd.: forward; rev.: reverse.
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3
Peter I, Graf C, Dummer R, Schaffner W, Greber UF, Hemmi S. A novel
attenuated replication-competent adenovirus for melanoma therapy. Gene
Ther 2003; 10: 530-539.
4
Pilder S, Logan J, Shenk T. Deletion of the gene encoding the adenovirus 5
early region 1b 21,000-molecular-weight polypeptide leads to degradation of
viral and host cell DNA. J Virol 1984; 52: 664-671.
5
Heise C, Hermiston T, Johnson L, Brooks G, Sampson-Johannes A, Williams
A et al. An adenovirus E1A mutant that demonstrates potent and selective
systemic anti-tumoral efficacy. Nat Med 2000; 6: 1134-1139.
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Hemmi S, Geertsen R, Mezzacasa A, Peter I, Dummer R. The presence of
human coxsackievirus and adenovirus receptor is associated with efficient
adenovirus-mediated transgene expression in human melanoma cell cultures.
Hum Gene Ther 1998; 9: 2363-2373.
7
Sirena D, Lilienfeld B, Eisenhut M, Kalin S, Boucke K, Beerli RR et al. The
Human Membrane Cofactor CD46 Is a Receptor for Species B Adenovirus
Serotype 3. J Virol 2004; 78: 4454-4462.
8
Havenga MJ, Lemckert AA, Ophorst OJ, van Meijer M, Germeraad WT,
Grimbergen J et al. Exploiting the natural diversity in adenovirus tropism for
therapy and prevention of disease. J Virol 2002; 76: 4612-4620.
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