j A i™ 1 1-1:' 
B«l 1*1 WII Us I 
%m\ 
Structure of the MSV DHFR-NEO genome. The derivation 
of the Moloney MSV vector backbone has been described in detail 
elsewhere 15 . Briefly, the sequences of the Moloney murine sarcoma 
virus genome that have been retained in the vector include the 
LTRs, sequences involved in the reverse transcription process and 
sequences necessary for the efficient encapsidation of the viral 
genome 21 (designated *). The S' splice site used in the generation 
of Moloney MLV env message is designated 5'ss. The retroviral 
gag-pol sequences have been replaced by a wild-type mouse cDNA 
encoding dihydrofolate reductase 23 . Because the Moloney MSV 
genome lacks the 3'ss signals necessary for generation of spliced 
tnv transcripts, sequences encoding the splicing signal have been 
re-introduced into the vector as shown. Adjacent to a 1.4-kb DNA 
fragment encoding neomycin and G4I8 resistance in bacterial and 
mammalian cells, respectively 23 (labelled neo), there is a small 
FnuDU fragment of pBR322 encoding the plasmid origin of repli- 
cation. The total sue of the MSV DHFR NEO genome is S.l kb. 
Restriction endonuclease cleavage sites are as shown. AAoIII docs 
not cleave the recombinant viral genome. The +2 line harbouring 
the genome produces a litre of >10 7 G4 1 8-resistant colonies per 
ml of culture fluid on 3T3 cells. The recombinant genome that is 
transmitted contains a deletion of a majority of DHFR sequences 
(C. Cepko, unpublished). 
IA>;I l(A*s oU ll£-‘ 
rate 
J-c 
I 1 
LTR 
Fig. 1. Diagram of the int* 
grated vector (proviral) 
N2. 0 to 1.5 and 3.0 to 3.8 
kb: Moloney murine leuke- 
mia vims sequences; 1.5 to 
3.0 kb box: Tn5 sequence 
containing the neo” gene 
Scale in kiobates (Bgl 1-Bam HI fragment 
from Tn5) (14), the hatched area is the coding sequence. LTR, long terminal repeat; 5*. the 
donor splice site at the 5' end; packaging sequence; restriction enzyme sites: S, Sac I; P, Pst 
1; E. Eco Rl; X, Xho 1; C. Cla I. 
LTR s' V 
_L 
_L 
OS 
J 
10 
IS 
x dt , $c lenct 
1395 
Recombinant DNA Research, Volume 12 
[193] 
