HHper-Fre* Retrovins Vector* 
B. 
A. 
PS I -2 CELLS 
TRANSFECT 
CELLS 
PLASMID CONTAINING 
SELECTABLE 
RETROVIRUS VECTOR 
i r 
2 DAYS LATER, 
HARVEST VIRUS 
AND INFECT 
PA12 CELLS 
AMPHOTROPIC VECTOR 
PROOUCING CLONES 
CLONE VECTOR-INFECTED CELLS 
IN SELECTIVE MEDIUM 
PA12 CELLS 
TRANSFECT 
CELLS 
y 
2 DAYS LATER, 
HARVEST VIRUS 
AND INFECT 
PS I -2 CELLS 
BCOTROPIC VECTOR 
PROOUCING CLONES 
TEST CLONES FOR: 
1. VECTOR TITER 
2. UNREARRANGH) VECTOR 
3. ABSENCE OF HELPER VIRUS 
n*.i. Genemion of vector-producing cell lines. 
limitation that many pieces and rearranged 
copies of vector DNA are generally integrated 
after transfection, and the resultant virus may 
be a mixture of desired and rearranged 
viruses. We have documented this problem 
using a virus containing hypoxanthine-gua- 
nine phosphoribosyllransferase (HPRT) and 
a rat growth hormone minigene (9). Virus 
rescued from transfected cells was able to 
convert HPRT' cells to HPRT*, but not all 
HPRT* colonies made growth hormone. 
We found, however, if cell lines were 
made that contained a single integrated copy 
of the HPRT-growth-hormone-virus as a re- 
sult of virus infection, that virus rescued from 
such cells invariably induced growth hormone 
production in infected cells selected for 
expression of HPRT (9). Both genes were 
coordinate^ transferred, indicating that the 
virus produced was not grossly rearranged. 
DNA and RNA analysis confirmed this inter- 
pretation (9). Thus a second strategy for 
production of helper-free virus involves trans- 
fection of the retrovirus packaging construct 
DNA into cells infected with the viral vector 
(4). Helper-free virus produced in this way 
will be more homogeneous than that produced 
following transfection of the vector DNA. 
However, this strategy is time consuming, as 
one must first isolate vector-infected cells, 
introduce the retrovirus packaging construct 
DNA into these cells by cotransfection with a 
selectable marker, and then isolate and screen 
transfected clones for production of helper- 
free virus vector. 
Direct infection of retrovirus packaging 
cells with the viral vector would considerably 
shorten this process and still lead to the pro- 
duction of homogeneous virus. This should be 
possible through the use of retrovirus packag- 
ing cell lines that can produce viruses having 
different host ranges. The ability of a retrovi- 
Recombinant DNA Research, Volume 12 
[361] 
