Pstl-BamHl IRAP fragment was then inserted into MFG, digested with Ncol and 
BamHl, using a synthetic adapter CATGGAAATCTGCA 
CTTTAG 
containing a 5' Ncol site and a 3' Pstl site; the initiation codon of IRAP is underlined. 
The resulting plasmid, MFG-IRAP (see Fig. 2) contains the entire coding sequence of 
IRAP. 
Construction of MFG-TK: To introduce the herpes simplex tk gene into MFG, 5' 
Ncol and 3' BamHl sites were introduced into the tk gene by PCR. A 5' primer was 
used that converted the two base pairs immediately 5' to the ATG of tk to CC, resulting 
in a Ncol site. The 3' oligonucleotide pirmer was used that converted a single A 
nucleotide to a T nucleotide 29 bases downstream from the stop codon of tk to create a 
BamHl site. The PCR reaction was performed using Vent polymerase. The resulting 
1151 base pair fragment was digested with Ncol and BamHl and inserted into MFG at 
the Ncol and BamHl sites to give MFG-tk (see Fig. 3). 
Construction of DFG-IRAP-tk: MFG-tk was digested with Ncol and Dralll and 
the large fragment isolated. Dralll digests MFG at a site 172 base pairs upstream from 
the Ncol site. The pCITE plasmid (Novagen) containing the encephalomyocarditis virus 
internal ribosome entry site (pCITE: Novagen) was digested with EcoRl, BamHl 
tinkered, and then digested with BamHl and Ncol. MFG-IRAP was digested with 
Drain and BamHl and the 737 base pair IRAP-containing fragment isolated. The 
MFG-tk fragment, IRAP and the 600 base pair IRES BamHl to Ncol fragment were 
ligated in a three part ligation to give DFG-IRAP-tk (see Fig. 1). 
Generation of high titer producer lines: The CRIP packaging line used for the 
experiments was checked by Microbiologic Associates for the presence of certain 
contaminating biological agents prior to use. The MFG-IRAP and MFG-tk vectors were 
cotransfected into CRIP packaging cells to generate high titer producers. lOg of the 
vector was cotransfected with 0.5 g of pSV2neo and the cells selected in G418 (.75 
mg/ml). Single colonies were isolated and expanded. The supernatants from the clones 
were screened for high titer virus. For MFG-IRAP, two mis of supernatant from each of 
the expanded colonies was added with 8/ig of polybrene/ml to a 10 cm2 plate of NIH 
3T3 cells approximately 50% confluent. 72 hours post infection the supernatants from 
the infected cells were assayed for IRAP production using an ELISA assay (R&D 
Systems). The producer able to confer the highest level of IRAP expression to 3T3 cells 
500ng/10^ cells/day was then assayed for presence of replication competent virus as 
described below. For MFG-tK, 2 mis of supernatant from each of the expanded, G418 
resistant colonies was added to 143tk- cells with 8 g/ml of polybrene. 48 hours 
post-infection the infected cells were split 1:10 into media containing HAT media and 
the number of tk+ colonies determined two weeks post-infection. The highest producer 
(3xl0^/ml) was then checked for replication competent virus. A similar method is 
currently being used to generate a high titer DFG-IRAP-tk producer. If the titer of 
DFG-IRAP-tk needs to be increased, multiple rounds of infection of the CRIP-DFG 
Recombinant DNA Research, Volume 19 
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