Miller «aL 
much still to be learned about proper virus 
construction to avoid these problems and pro- 
vide efficient transfer and expression of 
desired genes. Here we describe an improved 
method for rapidly making cell lines that 
produce retrovirus vectors containing domi- 
nant selectable markers. We have also ana- 
lyzed the factors which lead to helper virus 
production using some vectors, and describe 
methods to avoid this problem. 
MATERIALS AND METHODS 
Cells and Viruses. Cultured cells were 
grown in Dulbecco-Vogt modified Eagle 
medium with high glucose (4.5 g/liter) and 
10% fetal bovine serum in a 10% C0 2 atmo- 
sphere at 37°C, with the exception that Psi-2 
cells were grown in 10% calf serum instead of 
fetal bovine serum. Serum requirements for 
the cell lines were strict, and substitution 
adversely affected virus production from the 
cells. Cells were free of Mycoplasma, as 
determined by using the DNA stain Hoechst 
33258 (25). N1H 3T3 (TK") cells (7) produc- 
ing Moloney murine leukemia virus (Mo- 
MLV) or amphotropic MLV (AM-MLV) 
were made by transfection of virus DNA clone 
pMLV-K (26) or pAM (4), respectively, into 
the cells followed by passage of the cell for two 
weeks to allow viral spread to all cells. The 
PA 12 amphotropic retrovirus packaging cell 
line (4) and the Psi-2 ecotropic retrovirus 
packaging cell line (2) have been described. 
Virus Rescue Following Transfec- 
tion. Retrovirus packaging cell lines were 
plated at 5 x 10 s cells/60-mm dish on day 1. 
On day 2, the culture medium was replaced 
with 4 ml fresh medium, and 10 ug viral 
plasmid DNA (without carrier DNA) was 
transfected onto the cells by using the calcium 
phosphate precipitation procedure (27, 28). 
Briefly, a DNA-CaClj solution was prepared 
containing DNA at 50 ug/ml and CaCl 2 at 
250 mM and was added dropwise to an equal 
volume of freshly prepared buffer (27) con- 
taining 50 mM HEPES-NaOH, pH 7.1, 
250 mM NaCl, and 1.5 mM Na 2 HP0 4 - 
NaH 2 P0 4 , pH 7.0. After 30 min at room 
temperature, 0.4-mI aliquots of the mixture 
were added to each plate of cells. Cells were 
exposed to the DNA precipitate until day 3 
when the medium was aspirated and fresh 
medium was added. On day 4 the medium was 
removed, centrifuged at 3000 g for 5 min to 
remove cells and debris, and aliquots were 
used to infect cells. 
Virus Infection and Assay. Cells to be 
infected were seeded at 10 5 cells/60-mm dish. 
The next day the medium was replaced with 
fresh medium containing 4 ugfm\ polybrene 
and aliquots of virus were added to the cells. 
Sixteen hours after infection, the medium was 
replaced with selective medium containing 
10' 7 M methotrexate and dialyzed serum for 
assay of the DHFR-virus or 2 mg/ml G418 
(about 50% active) for assay of the Neo- 
virus. For generation of vector-infected pack- 
aging cell clones (Figure 1), retrovirus pack- 
aging cells were infected with 10—100 /xl virus 
rescued after transfection, and 16 h later were 
treated with trypsin and divided 1:10 into 
100-mm dishes containing selective medium. 
Resultant colonies were isolated by using 
cloning rings. The infection protocol was mod- 
ified for the experiments shown in Table 2; the 
cells were plated initially at 5 x 10 5 /60-mm 
dish, and 16 h after infection the cells were 
treated with trypsin and divided 1:10 into 
medium containing selective agents. Helper 
virus was monitored using the S*L“ assay as 
previously described (4). 
RESULTS 
Several strategies have been used for the 
generation of helper virus-free retrovirus vec- 
tor. One strategy involves transfection of the 
retrovirus vector into a retrovirus packaging 
cell line, followed by selection for cells con- 
taining the vector (2, 18, 20, 21). RNA tran- 
scribed from the transfected vector DNA is 
packaged into virions by proteins made by the 
packaging cells. This strategy suffers from the 
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Recombinant DNA Research, Volume 12 
