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Biochemistry 
Self-inactivating retroviral vectors designed for transfer of whole 
genes into mammalian cells 
(neomycin-resistance select ion /c-/oi/gene therapy! 
Sheau-Fung Yu*. Thomas von Ruden*. Philip W. Kantoff*. Christa Garber*, Miri Seiberg*. 
Ulrich Ruther*. W. French Anderson + . Erwin F. Wagner*, and Eli Gilboa‘5 
'Department of Molecular Biology. Princeton University. Princeton. NJ 08544: ^Laboratory of Molecular Hematology. National Heart. Lung, and Blood 
Institute. National Institutes of Health. Bcthcsda. MD 20892; and ^European Molecular Biology Laboratory . Mcycrhofstrassc I. 
D-6900 Heidelberg. Federal Republic of Germany 
Communicated hy Marshall Nirenherg. January J. IVN6 
ABSTRACT A retrovirus-derived vector called self-inac- 
tivating (SIN) vector was designed for the transduction of whole 
genes into mammalian cells. SIN vectors contain a deletion of 
299 base pairs in the 3' long terminal repeat (LTR), which 
includes sequences encoding the enhancer and promoter func- 
tions. When viruses derived from such vectors were used to 
infect NIH 3T3 cells, the deletion was transferred to the S' 
LTR, resulting in the transcriptional inactivation of the 
provirus in the infected cell. Introduction of a hybrid gene 
(human metallothionein-promoted c -fos) into cells via a SIN 
vector was not associated with rearrangements and led to the 
formation of an authentic mRNA transcript, which in some 
cases was induced by cadmium. SIN vectors should be partic- 
ularly useful in gene transfer experiments designed to study the 
regulated expression of genes in mammalian cells. Absence of 
enhancer and promoter sequences in both LTRs of the inte- 
grated provirus should also minimize the possibility of activat- 
ing cellular oncogenes and may provide a safer alternative to be 
used in human gene therapy. 
Retroviruses are now being used as an efficient delivery 
system for transferring genes into tissue culture cells (1-6) 
and into intact animals (7-11). Replication of retroviruses is 
dependent on an efficient process unique to this group of 
viruses. This process involves the insertion of the viral 
genome into the chromosome of the infected cell, from which 
the viral genes are constitutively expressed, in most cases 
without measurable effect on the viability of the infected cells 
(for a review, see ref. 12). This efficient process is the basis 
for using retroviruses as vectors for gene transfer. An 
expendable portion of the viral genome (containing functions 
that can be complemented in trims) is replaced with a foreign 
gene. The result is an efficient gene-transfer system in which 
a large fraction of recipient cells will have incorporated and 
will express the transduced gene. 
In this report, we describe the construction of a retroviral 
vector that transmits a nonselected transduced gene in a 
stable manner. With this type of vector, the viral enhancer 
and promoter sequences are lost upon integration into the 
chromosome of the target cell, allowing nonviral regulation of 
the expression of the transduced gene. The elimination of the 
enhancer and promoter sequences from both long terminal 
repeats (LTRs) in the target cell is also useful in the design of 
safe vectors for potential use in human gene therapy (13). 
because they may reduce substantially the possible activation 
of cellular oncogenes. Since the loss of enhancer and pro- 
moter sequences is a consequence of the mechanism of 
replication of retroviruses, resulting in the transcriptional 
The publication costs of this article were defrayed in pan by page charge 
payment. This aniclc must therefore be hereby marked "advertisement" 
in accordance with IK U.S.C. §1734 solely to indicate this fact. 
inactivation of the provirus in the target cell, this type of 
vector is called a SIN (.self-i/iactivating) vector. 
MATERIALS AND METHODS 
Derivation of Recombinant Virus. Vector DNA was intro- 
duced into d/2 cells (14) by calcium phosphate-mediated DNA 
transfection (15). and single colonies were isolated by selec- 
tion with G418 (1 mg/ml) (16. 17). and expanded to cell lines, 
and 1.0 ml of the supernatant containing =10* colony-forming 
units of nerZ-expressing viruses per ml was used to infect 
1. 0-2.0 x 10 5 NIH 3T3 cells. Productively infected cells were 
isolated in selective medium. 
Southern Blotting. Genomic DNA was isolated, digested 
with A flie I. subjected to electrophoresis in 1 9c agarose gels 
(18). blotted to Biodyne A paper, and hybridized with a 
,: P-labe!ed probe. Each DNA preparation was derived from 
NIH 3T3 cells infected with a virus preparation originated 
from an independently transfected d»2 cell. 
RNA Blotting. RNA was isolated from NIH 3T3-infected 
cells using 0.1 7c Nonidet P-40 in 10 mM TrisHCl. pH 7. 5/0. 2 
M NaCI/5 mM MgCK, and nuclei were removed by centrif- 
ugation at 1000 x g for 5 min. NaDodS0 4 was added to the 
supernatant, extracted four times with phenol/chloroform 
(1:1) and twice with chloroform, and precipitated with 
ethanol. Poly(A)-containing cytoplasmic RNA was isolated 
by oligo(dT) cellulose chromatography and electrophoresed 
in \% formaldehyde/agarose gels (19). transferred to 
Biodync A paper, and hybridized with a 52 P-labeled probe. 
RESULTS 
Structure of the SIN Vectors. The principle underlying the 
structure and function of the SIN vectors is illustrated in Fig. 
1. The integrated retroviral genome, called a provirus, is 
bound by two LTRs, which are composed of three regions: 
U3, R, and U5. The U3 sequence is present in the 3' end, R 
is on both ends, and U5 is at the 5' end of the viral RNA (Fig. 
1A). The integrated proviral genome is transcribed from LTR 
to LTR: the 5' end of the viral RNA corresponds to the 5' end 
of the R region and the 3' end of the viral RNA, which is 
polyadenylylated, corresponds to the 3' end of the R region 
at the other end of the genome (Fig. 1A). The enhancer and 
promoter sequences are encoded within the U3 region of the 
two viral LTRs. However, they function only when present 
in the 5' LTR. It is not clear why the same sequences are 
inactive in the 3' LTR. It is a consequence of the mechanism 
of replication of retroviruses that the U3 region in the 3' LTR 
Abbreviations: LTR. long terminal repeat: Mo-MuLV. Moloney 
murine leukemia virus: SV40. simian virus 40: MT. metallothionein: 
TK. thymidine kinase: bp. base pairts): kb. kilobase(s). 
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