HEPATOCELLULAR TRANSPLANTATION AND TARGETING GENETIC MARKERS TO HEPATIC CELLS 
Figure 3 . 
RNA tRNA 
VIRAL CAP binding PACKAGING 
enhancers site site SIGNAL 
ISD / ATG 
ORIGIN OF 
SECOND STRAND POLY (A ) 
DNA SYNTHESIS SITE 
pAM 
Clal 
pPAM 
pPAM2 
pPAM3 
p.„-i4 
CKl 
Clal 
Sau3AI 
SA TAG 
I- 1 
^ gag. pol, ar 
Bal I Pat I 
SO ATG 
( Bal I) 
1 = ^ 
SA tag 
V Lr 
gag. pol, arw /' 
Pat I 
SO ATG 
SV40 (Balli 
POLY(A) t oa* 1 1 
SITE 
SA TAG 
I — I I I sag. pol. anv 
I _ a lH / 0 
Pat I 
SO ATG 
BamHI (Bell) 
SA TAG 
I 
gag. pol, anv 
Patl 
CAP SO ATG 
SA tag 
gag. pol. aov 
( Bell) 
TAG 
Sau3AI Sau3Al Patl 
( Bel I ) 
N2 
LNL6 
MoMLV 
MoMLV 
^CJQ ATG 
NEO 
pA 
MoMSV 
MoMLV 
MoMLV 
1 kb 
Figure 3A 
Packaging constructs are depicted without surrounding plasmid sequences. Large, open boxes represent retroviral LTRs; small, 
open boxes represent other retroviral sequences; and hatched boxes denote simian virus 40 sequences. Landmark restriction sites are shown, 
but all of the sites for a given enzyme are not necessarily shown. The plasmid containing a recombinant amphotropic helper virus (pAM) and 
the derivative of this virus in which the packaging sign^ has-been removed (pPAM) have been described before (15). Important features for 
retrovirus replication are noted above the pAM construct. The construct pPAM2 was made from pPAM by replacement of the 3' LTR with 
the late polyadenylation signal from simian virus 40. isolated as a 237-base-pair BamHI to Bell fragment. The end of the retroviral genome 
was cleaved with Rsal at position 7762 (25). which cuts Just upstream of the termination codon of env, and a synthetic oligonucleotide was 
added to duplicate the part of env that was removed. This oligonucleotide also contained a BamHI site downstream of the terminator codon 
for env for addition of the simian virus 40 fragment containing a polyadenylation signal. pPAM3 was made from pPAM2 by removing viral 
sequences 5' of the viral enhancers in the 5' LTR by using an Jflu3AI site at position -352 (25). In addition to the deletions in pPAM3, pPAM4 
has a deletion which removes the tRNA-binding site and 3' portion of the 5' LTR while preserving the splice donor site. This deletion was 
made by cleavage of the LTR at an Smal site at position 28 (25) in the R region of the LTR, addition of a BamHI linker, and linkage to an 
5au3AI site at position 161 (25) {BamHI and Sau3Al leave complementary 5' extensions). The constructs are all carried in pBR322. pAM. 
pPAM, and pPAM2 are inserted in pBR322 in place of the Tc' gene between the Clal and P\'ull sites. The Clal sites remain, but the sites at 
the other ends of the constructs were destroyed during attachment to the /’vwll site of pBR322. pPAM3 and pPAM4 were inserted in place 
of the Tc' gene between BamHI and Pvull. Only the Sau3Al site remains at the 5' end of the construct, and the sites at the 3' end have been 
destroyed, kb, Kilobasc; SD, splice donor; SA, splice acceptor. From Miller and Buttimore, 1986. Figure 3B 
Structure of N2 and LNL6 retroviral constructs. From Miller and Rossman, 1989. 
Recombinant DNA Research, Volume 14 
[811] 
