Somatic Cell and Molecular Genetics. Vol. 12. Mo. 2. 1986. pp. 175-183 
Factors Involved in Production of Helper Virus-Free 
Retrovirus Vectors 
A. Dusty Miller, 1 David R. Trauber, u and Carol Buttimore' 
‘The Fred Hutchinson Cancer Research Center. 1124 Columbia Street. Seattle. Washington 98104; and * Laboratory 
of Molecular Hematology. Motional Heart. Lung, and Blood Institute, Bethesda. Maryland 20892. 
Received 8 November 1985 — Final 6 December 1985 
Abstract — Retrovirus vectors allow efficient transfer of genetic material into cells. We 
describe an improved method for making cell lines which secrete broad host range retrovirus 
vectors in the absence of helper virus. This method was used to make virus-producing cell lines 
from several retrovirus vector constructions that encode dominant selectable markers. Virus 
titers from such lines exceeded l(f colony-forming units per milliliter of medium exposed to 
the cells. Cell lines that secreted certain vectors remained free of helper virus, while cell lines 
made using other vectors always secreted helper virus. Secretion of helper virus apparently 
depended on recombination between vector and the retrovirus packaging system, and factors 
Involved in this event were investigated. 
INTRODUCTION 
Retrovirus vectors have proven useful for 
efficient transfer of genetic information into 
cells in culture and into animals. Selectable 
(1-7), nonselectable (8-10), and inducible 
genes (9, 11) have been transferred in culture. 
We and others have demonstrated transfer of 
selectable genes into embryos (12, 13) and 
into the hemopoietic cells of mice (14-17). 
The development of cell lines for packaging of 
replication defective viruses in the absence of 
helper virus (2, 4, 18-21) has enhanced the 
utility of retroviruses for gene transfer. In 
animals, the effects of helper virus replication, 
including disease, obscure the effects of vec- 
tor-encoded products. In the absence of helper 
virus, there is no spread of the vector following 
the initial infection, which simplifies experi- 
ment interpretation. Retrovirus vectors ap- 
pear to be the vehicle of choice for initial 
attempts at gene therapy in humans (22), and 
these techniques will require vectors that are 
not contaminated with helper virus. 
The design of retrovirus vectors for gene 
transfer is not straightforward. Certain poorly 
defined sequences in viral constructs can be 
deleted or rearranged or can act to inhibit 
virus replication (I, 23, and unpublished 
results). Difficulties in expressing two genes 
in a vector have been reported (24), and the 
ability to transfer two genes is important in 
order to transfer nonselectable genes linked to 
selectable markers. In addition, we show here 
that it is difficult to make helper-free virus 
from some vectors because of interactions 
between vector and packaging cell lines which 
lead to helper virus production. Thus, there is 
’Present address: Division of Hematology, Downsute Medical Center, State University of New York, 450 Clarkson 
Avenue, Brooklyn, New York 1 1203. 
175 
0740-7750/14/0300-01 73S05.00/0© 19*4 Plenum Publishing Corporation 
Recombinant DNA Research, Volume 12 
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