to the Narl site at position 1035 to increase the packaging efficiency of the vector. The 
vector also contains a 390 base pair MLV fragment from the Ndel (5402) to Xbal 
(5794) sites which contains the splice acceptor. An 18 base pair oligonucleotide 
containing Ncol and BamHl sites directly follows the synthetic oligonucleotide which 
allows for insertion of cDNAs with compatible ends. The Ncol site is positioned so that 
the initiation codon of the inserted gene is fused to the initiation ATG of the viral env 
gene. Thus the inserted gene is expressed from a spliced message that resembles the 
normal env message of MoMLV. The MoMLV LTR drives expression of the mRNA 
which contains a 5' untranslated region identical to the normal env message followed 
directly by the coding region of the inserted gene. In the MFG vector, there is an 8 base 
pair SacII linker inserted just downstream of the gag ATG at a Haelll site which shifts 
the gag reading frame out of frame. 
Construction of the MFG vector; The retroviral vector was constructed in several 
steps. First a three part ligation was performed where a Xhol (position 1035; originally 
a Narl site) to Ndel (position 2295 in pBR322) 5' LTR fragment from a half gag vector, 
and a BamHl (originally a Clal site at position 7675) to Ndel (position 2295 in 
pBR322) 3' LTR fragment from the pEM vector were ligated to a Xhol to BamHl 
histone promoter filler fragment. Second, the Ndel site (position 2295 in pBR322) was 
destroyed by partial digestion with Ndel followed by a klenow fill in reaction. The Xhol 
site (originally a Narl site at position 1035 in MoMLV) was Ndel tinkered. Third, the 
large Ndel to BamHl fragment from the intermediate vector was then ligated to a Ndel 
(position 5402 in MoMLV) to Xbal (5794) fragment from MOV-9, containing the splice 
acceptor sequence, and a synthetic adapter fragment containing a Ncol site positioned so 
that the ATG within the Ncol recognition site is the initiation codon of the env gene. 
Adapter sequence: CATG ACTGCCATGGCGCG 
TGACGGTACCGCGCCTAG 
Cloning of IRAP: A human monocyte cDNA library in lambda gtlO was purchase 
from Clontech (catalog number HL1036a). The library was derived from the human 
monocyte cell line U937 which had been stimulated with 10 nM phorbol 12-myristate 
13-acetate for 48 hours before isolation of the mRNA. The library was screened for 
IRAP clones via a standard plaque lift assays from agar plates to nitrocellulose filters. 
Two phage clones with EcoRl-flanking sites were isolated and subcloned into the EcoRl 
sites of pUC18 and shown to have identical restriction maps. One of the clones was then 
sequenced and amplified by PCR. The resulting IRAP insert had the following structure: 
a 5' Hindlll site followed immediately by bp 1, the entire IRAP protein coding sequence, 
3' flanking DNA from bp 543-577, followed immediately by a 3' Hindlll site. This insert 
W 21 S subcloned in the Hindlll site of pSV2. 
Construction of MFG-IRAP: The cDNA for human IRAP was inserted into the 
retroviral vector MFG by first BamHl linkering the 3' Hindlll site downstream of the 
stop coding in the IRAP gene followed by digestion with Pstl and BamHl. The 
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Recombinant DNA Research, Volume 19 
