B. 1 Construction of MFG 
Subsequent studies with the N2-SV-GC vector were not encouraging that expression of 
the transgene was robust. In an effort to overcome the failure to achieve sustained 
expression with earlier vectors, we obtained the MFG vector to test its ability to express 
the GC gene in hematopoietic cells. The MFG retroviral vector was originally constructed 
by Dr. Robbins in Dr. Richard Mulligan's laboratory. Dr. Robbins provided the plasmid to 
us. The features of this vector are that the GC cDNA is transcribed by the retroviral LTR 
and the start codon of the GC cDNA was placed at the start codon of the deleted envelope 
protein gene. No internal promoter or dominant selectable marker is included in the 
construct. The structure of MFG is shown in Figure 1. 
The MFG vector contains 1035 base pairs of the gag sequence from MMLV to increase the 
encapsulation of recombinant genomes in the packaging cell lines, and 350 base pairs derived 
from MOV-9 which contains the splice acceptor sequence and transcriptional start. An 18 
base pair oligonucleotide containing Ncol and BamHI sites directly follows the MOV-9 
sequence and allows for the convenient insertion of genes with compatible sites. The Nco 
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 MMLV LTR controls transcription 
and the resulting mRNA contains the authentic 5' untranslated region of the native gag 
transcript followed directly by the open reading frame of the inserted gene. 
The MFG 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 BamHI (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 BamH2 histone H4 pormoter filler fragment. Second, the 
Ndel site (position 2295 in pBR322) was then destroyed in the pBR322 backbone of the H4 
intermediate and the Xhol site at position 1035 then Ndel linkered. Third, the large Ndel 
to BamHI fragment from the intermediate factor was then ligated to a Ndel (position 5402) 
to Xbal (5764) fragment from MOV-9, containing the splice acceptor sequence, and a synthetic 
adapter fragment containing Xbal and Bamhl overhangs (shown below) to give MFG. The 
synthetic adapter contains a Ncol site positioned so that the ATG within the Ncol 
recognition site is the initiation codon of the env gene. BamHI and the large fragment 
I 
Adapter sequence: CTAGACTGCCATGGCGCG 
TGACGGTACCGCGCCTAG 
B. 2 Construction of MFG-GC 
) 
To clone into the MFG vector, we created an Nco I site in the position of the start 
codon (ATG) of the GC cDNA using PCR. The sense primer (5' -CCACCATGGCTGGCAGCCTC-3' ) was 
made with a one base pair mismatch to create the Nco I site. The antisense primer (5'- , 
GTGTACTCTCATAGCGGCTG-3' ) was located down stream of a Hind III site. PCR was carried out 
using a thermal cycler (Perkin Elmer Cetus, Norwalk, CT). The product was cut with Nco I 
and Hind III and a 60 bp fragment was isolated using 4% NuSieve agarose gel (Rockland, ME). 
On the 3' side of the GC cDNA, an Eco RI fragment of GC cDNA was isolated and the terminus 
was filled in with dNTP's by the Klenow fragment to create a blunt end. A Bel I linker was 
ligated in the usual manner and digested with Hind III and Bel I. The 1.7 Kb Hind III/Bcl 
I fragment was isolated from 1% low melting point agarose gel. The 60 bp Nco I/Hind III 
fragment and 1.7 Kb Hind III/Bcl I fragment were ligated to Nco I/Bam HI site in the MFG 1 
vector. The authenticity of this construct was confirmed by DNA sequencing and revealed 
no PCR errors or cloning artifacts. The provirus structure is shown in Figure £. The ; 
sequence of the entire MFG-GC vector was performed by Lark Sequencing Technologies, Inc. 
in compliance with directives issued by the EPA/FDA. The sequencing strategy and results 1 
are included in the appendix and on disk BA.l.SEQ. 
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Recombinant DNA Research, Volume 17 
