100 
- 80 
■ ♦ 11-3 
E3 - n--3 
20 
Id 
II 
10 * 10 * 10 4 10 * 10 * 
Virua IH*r 
Axs Bpxs C 5 p Fig. 2 (left I. Tiler cflect\ upon efficiency of 
hone marrow infection. Indicated liters were 
. ^ obtained from clonal populations of various 
N2-prixJucing cells as tabulated in Table I. 
Culture conditions were as described in the 
legend to Table I. Fig. 3 fright). Southern 
blots of DNA prepared from individual pri- 
mary spleen foci and a whole, reconstituted 
mm spleen. Bone marrow cells were co-cultured. 
% as described in the legend to Table I. for 24 
hours, with F-5B cells producing N2 at a titer 
* • of 2 x 10* cfu/ml. DNA from two individually 
• . isolated 10-day spleen foci obtained from in- 
* feclions done at different times (A and B) and 
_ from a long-term reconstituted spleen (C) was 
prepared as described in Table I. The spleen 
DNA was prepared from a mouse 4 months after the lethally irradiated animal received 5x10* 
infected bone marrow cells. For blots A. B. and C equal amounts of DNA (30 p.g per lane) were 
digested with restriction enzymes and then subjected to electrophoresis through 0.7 percent 
agarose gels. After electrophoresis, the gels were blotted ( 18) onto Biotrans filler membranes 
according to the suppliers' (ICN) instructions. Hybridizations were as described in the legend to 
Table I. with films exposed for 5 days. Enzyme digestions were with Xho I (X). Sac I (S). and 
Pst I (P). Large arrows indicate the position of the 3.2-kb Sac I fragment (Fig. 1 1: small arrows 
indicate the position of the l).9-kh Pst I fragment (Fig. I). 
Table I. Spleen focus analyses from bone marrow infections. N2. a Moloney-based retroviral vector containing the neo" gene, was transfected 
using calcium phosphate 1 ' into 'P2 cells at 20 wg per 5 * 10' cells per tissue culture plate (100 mm). Permanently transfected G4I8* clones were 
isolated after 10 to 14 days selection, then individually expanded and the number of virus particles conferring G4IX resistance to 3T3 cells was de- 
termined. The individual cell lines were grown to confluence at 37*C in 5 percent CO; in air at 100 percent humidity on tissue culture plates ( 100 
mm) in 10 ml of NIH 3T3 medium defined as DMEM (Dubecco minimal essential medium) (Biofluids. No. 104) plus 10 percent (by volume) 
defined fetal bovine serum (Hyclone) plus freshly added L-glutamme (300 ug/ml). The medium was changed and then removed 24 hours later: it 
was centrifuged to remove floating cells, and the supernatant (containing viral particles) was frozen until the time of titration. The 3T3 cells for ti- 
tration were plated (5 * Iff* cells per 60-mm plate) in 4 ml of 3T3 medium, and 18 to 24 hours later the medium was removed and replaced with I 
ml of serial diluted thawed viral supernatant containing Polybrene (8 wg/ml) to increase virus adsorption. Plates were rocked every 15 minutes for 
2 hours at which point 4 ml of fresh 3T3 medium was added. About 48 hours after infection the culture fluid was replaced with fresh 3T3 medium 
containing G4 18 ( I mg/ml). From 10 to 12 days later, plates were scored for G4 1 8* colonies both by microscopic counting and by methylene blue 
staining. Bone marrow was isolated from female DBA/2J (of NFS/N) mice (8 to 12 weeks of age). It was flushed from both femurs with a-medium 
(AM EM. Biofluids. No. 109). Single cell suspensions were made, cell counts were determined, and 5 x 10* cells in a volume of less than I ml 
were added to a 100-mm culture plate containing a confluent monolayer of virus-producing 'P2 cells. The ^2 cells (2 x 10*) had been seeded into 
10 ml of 3T7 medium 48 hours earlier. Fresh 3T3 medium was added 24 hours before the addition of bone marrow cells. Individually isolated 'P2 
clones F-5B. E-IB. E-IA. E-4A. and F-2B were used to produce N2 vector at the indicated titers. Titers of 2 x 10' and 6 x 10' cfu/ml were 
achieved by plating the F-5B cell line at 1/10 and 1/3 the usual density. Polybrene was added to the cultures at a final concentration of 4 ml. The 
concentration of IL-3. when added, was 20 U/ml. Purified IL-3 (7) had a specific activity of 0.05 ng/unit: it was diluted 1 : 100 into RPMI I W0 with 
10 percent fetal bovine scrum, filter sterilized, and kept frozen until use. Cells were cultured in 10 ml of 3T3 medium plus penicillin ( 100 U/ml) and 
streptomycin ( 100 wg/ml) at 37*C in a 100 percent humidified atmosphere of 5 percent CO; in air. After the indicated times, bone marrow cells 
were recovered by gentle pipetting, and the plate was rinsed with 5 ml of a-medium. The pooled cells were sedimented and resuspended in o-me- 
dium at a final cell density of 5 * IO*/ml. A cell suspension of I x 10* cells (0.2 ml) was injected into the tail vein of each of several syngeneic 10- 
week-old mice that had been irradiated with 900 rad from a "’Cs source 2 to 3 hours earlier. Ten days after injection of the bone marrow cells, 
spleens were dissected and individual foci were isolated. Celt suspensions were made of each focus and DNA was prepared (16). DNA dot blot 
analysis of the individual foci (12 ug of DNA) was performed as described (17) with a nick-translated (Nick-Translation Kit. BRL) Hind lll-Bgl II 
fragment isolated from pNco (P-L Biochemicals). Ten positive foci were further analyzed by Southern blot analysis (18). and in all cases the 
presence of DNA of the expected restriction pattern was confirmed. The 49 foci analyzed for the * IL-3 24-hour data point were from a number of 
different animals infected in each of five independent experiments. The IS foci analyzed for the + IL-3 48-hour point were from three animals 
infected in one experiment. 
No IL-3 With IL-3 
Cell 
line 
Con- 
flu- 
ence 
(*) 
Co-cul- 
tiva- 
tion 
(hr) 
Initial 
titer 
Mouse 
strain 
Spleens 
studied 
TouJ 
foci 
Aver- 
se 
foci/ 
spleen 
Foci 
anal- 
yzed 
(A0 
DNA 
posi- 
tive 
(A0 
DNA 
posi- 
tive 
foci 
(%) 
Spleens 
studied 
Total 
foci 
Aver- 
age 
foci/ 
spleen 
Foci 
anal- 
yzed 
(A0 
DNA 
posi- 
tive 
(A0 
DNA 
posi- 
tive 
foci 
(%) 
F-5B 
100 
24 
1.2 * 10* 
DBA 
2 
10 
5.0 
9 
7 
78 
8 
94 
11.8 
49 
42 
86 
100 
48 
2 x 10* 
DBA 
1 
8 
8.0 
8 
6 
75 
8 
71 
8.9 
18 
14 
78 
F-5B 
100 
24 
1.0 x 10* 
NFS 
4 
38 
9.5 
23 
17 
74 
F-5B 
30 
24 
6 x 10' 
DBA 
8 
8 
100 
10 
24 
2 x 10' 
DBA 
16 
13 
81 
E-IB 
too 
24 
2 x 10' 
DBA 
1 
10 
10.0 
8 
5 
63 
2 
19 
9.5 
17 
15 
88 
E-IA 
100 
24 
6 x 10* 
DBA 
6 
65 
10 8 
38 
15 
39 
3 
32 
10.7 
30 
14 
47 
E-»A 
100 
24 
l.l x 10' 
DBA 
4 
31 
7.8 
22 
0 
0 
4 
24 
6.0 
23 
0 
0 
F-2B 
100 
24 
8 x 10-' 
DBA 
3 
19 
6.3 
12 
0 
0 
4 
40 
10.0 
16 
0 
0 
Totals 
17 
143 
8.4 
97 
33 
318 
9.6 
200 
I w* 
SCIENC E. vtiL 230 
Recombinant DNA Research, Volume 12 
[237] 
