Stem Cells, Vol. 17, No. 4, 226-232,
July 1999
© 1999 AlphaMed Press
Green Fluorescent Protein as a Selectable Marker of Retrovirally Transduced Hematopoietic Progenitors
Akihiro Kumea,
Motohiro Hashiyamab,
Toshio Sudab,
Keiya Ozawaa,c
a Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan;
b Department of Cell Differentiation, Institute of Molecular Genetics and Embryology, Kumamoto University School of Medicine, Kumamoto, Japan;
c Department of Hematology, Jichi Medical School, Tochigi, Japan
Key Words. Gene therapy • Green fluorescent
protein • Hematopoietic progenitor • Retrovirus
vector • Selectable marker • Flow
cytometry
Dr. Akihiro Kume, Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan.
 |
ABSTRACT
|
|---|
Recombinant retroviruses are most commonly used in hematopoietic
stem cell gene therapy trials, but gene transfer efficiency is still
inadequate with the present vectors. One approach for overcoming this
problem is to develop methods of selecting and enriching the
successfully transduced cells. We investigated the feasibility of
using the green fluorescent protein (GFP) gene as a selectable marker
of hematopoietic cells. When M1 murine leukemia cells were
electroporated with GFP expression vectors, a red-shifted mutant
(S65T) GFP showed several-fold greater fluorescence than the wild-type
GFP and generated readily detectable green light under control of SRa
or CAG promoter. We then inserted an SRa-S65T GFP cassette into the
MSCV retrovirus vector and established virus producer cells. Infection
of primary murine bone marrow cells resulted in a distinct population
with green fluorescence, which was separated by fluorescence-activated
cell sorting. The fractionated bright cells gave rise to fluorescent
spleen colonies in lethally irradiated mice, while the
fluorescence-negative cells yielded only dark colonies. These results
indicated that GFP is a faithful marker in gene transfer into
hematopoietic progenitor/stem cells, facilitating selection of the
transduced cells and tracking of their progeny in vivo. Stem
Cells 1999;17:226-232
|
Introduction
|
|---|
Since one of the key features of hematopoiesis is continuous
proliferation and differentiation into multilineages, precise
transgene distribution through cell division is a prerequisite for
long-term cure of most hematopoietic disorders by gene
transfer. Recombinant retroviruses can accomplish this task by
integrating transgenes into the host genome and are consequently
commonly used for gene transfer into hematopoietic cells. However,
retroviral transduction of hematopoietic stem cells (HSC) has been
hampered by inadequate gene transfer efficiency in larger animals,
including humans [1,
2]. Efforts to improve
vectors and transduction protocols are ongoing; however, reliable
means of monitoring in vivo transgene expression should also be
developed for quantitative evaluation.
In order to circumvent the efficiency problems, several selectable
markers have been incorporated into retrovirus vectors to enrich the
transduced cells. The markers have primarily been drug-resistance
genes, because the untransduced cells are eliminated by cytotoxic
drugs [3, 4]. However, longer ex vivo
manipulation of HSC would impair their immature phenotype, and
anticancer drug administration to patients without malignancy is a
matter of controversy. Recently, cell surface antigens such as CD24
and low-affinity nerve growth factor receptor have been shown to
facilitate rapid and efficient purification of transduced cells
[4-6]. One of the concerns with surface markers is
that antibody formation may be elicited against the antigens,
particularly when xenoantigens are employed.
We have evaluated the feasibility of using green fluorescent
protein (GFP) as a selectable marker in retroviral transduction. GFP
was cloned from the jellyfish Aequorea victoria, and the gene
encodes a relatively small cytoplasmic protein (27 kDa). Upon being
oxidized and cyclized, GFP molecules absorb blue light to emit green
fluorescence without substrates, cofactors, or other gene products
[7, 8]. This green light of native GFP or fusion
proteins can be detected by fluorescence microscopy or flow cytometry
in living cells without fixation. Thus, GFP has been welcomed with
much enthusiasm in the field of biological research for tracking cell
lineage and protein transport. For gene therapy technology, GFP may be
a useful marker of high-titer vector producers and successfully
transduced cells shortly after infection. The latter is particularly
important for stem cell gene therapy to maintain an immature phenotype
of the target cells. However, GFP expression has often been inadequate
for practical marking of mammalian cells [8]. We attempted to find better constructs for
GFP expression in hematopoietic cells with a limited number of copies
of the transgene.
|
Materials and
Methods
|
|---|
Plasmids
Wild-type (WT) GFP plasmids were provided by
Dr. S. Takagi (Nagoya University; Nagoya, Japan) [7]. A mutant GFP cDNA clone with an
amino acid substitution of threonine for serine at position 65 (S65T)
was a gift from Dr. R. Y. Tsien (University of California,
San Diego; La Jolla, CA) [9]. WT and S65T GFP cDNAs were cloned into
three mammalian expression vectors as follows. pRc/CMV is a
cytomegalovirus (CMV) promoter-containing vector (Invitrogen;
Groningen, The Netherlands); pMKITNeo has an SRa promoter (a gift from
Dr. K. Maruyama, Tokyo Medical and Dental University; Tokyo,
Japan) [10];
pCAGn-MCS-polyA has a CAG promoter in pSV2neo backbone (a gift from
Dr. T. Naruse, Kaketsuken; Kumamoto, Japan) [11, 12]. Besides the promoters mentioned above, all
three vectors contain the neomycin phosphotransferase (neo) gene
expression units, which allow G418-selection of the transfectants.
S65T GFP retroviruses were constructed with MSCV vectors which
originally had an internal murine phosphoglycerate kinase promoter
(PGK) driving neo gene (gifts from Dr. R. G. Hawley,
University of Toronto; Toronto, Canada) [13]. As illustrated in Figure 1, S65T GFP cDNA was inserted into the
multiple cloning site of MSCV2.1 for titration reference
(MSCV/S65Tneo). The modified MSCV vectors were constructed by
replacing the PGK-neo cassette in MSCV2.1 with CAG promoter-S65T GFP
(MSCV/CAG-S65T), and PGK-neo in MSCV2.2 with SRa promoter-S65T GFP
(MSCV/SRa-S65T).
View larger version (10K):
[in this window]
[in a new window]
|
Figure 1. Schematic representation of S65T GFP retroviral
vectors. MSCV/S65Tneo (top), MSCV/SRa-S65T (middle), and
MSCV/CAG-S65T (bottom). Each vector contains the S65T GFP gene (S65T)
in an MSCV backbone. LTR = long terminal repeat; PGK = murine
phosphoglycerate kinase promoter; neo = neomycin phosphotransferase
gene; SRa = SRa promoter containing the SV40 late-gene splice
junction; CAG = CAG promoter containing a chicken
b-actin/rabbit b-globin hybrid
intron.
|
|
Cells and Animals
M1 (American Type Culture Collection [ATCC] TIB-192; Manassas,
VA), NIH3T3 (ATCC CRL-1658) and GP+E86 (a gift from
Dr. A. Bank, Columbia University; New York, NY) cells were
maintained in Dulbecco's modified Eagle medium supplemented with
10% calf serum (JRH Biosciences; Lenexa, KS) [14]. Primary mouse bone marrow (BM) cells were
cultured in a-minimum essential medium containing 20% fetal calf serum
(CSL Ltd.; Parkville, Australia) and appropriate
cytokines. Fluorescence-activated cell sorting (FACS) was carried out
with a FACScan or a FACS Vantage (Becton-Dickinson; San Jose,
CA). C57BL/6 mice (eight weeks of age) were purchased from SLC
(Hamamatsu, Japan).
Transfection
Five million M1 cells in 0.5 ml phosphate-buffered saline were
transfected with 50 µg of each plasmid by electroporation at 280 V
and 960 µF using a Gene Pulser (Bio-Rad; Hercules, CA). After
electroporation, the cells were selected with 1 mg/ml G418 (Life
Technologies; Grand Island, NY) and the resistant M1 cells were
subjected to further analyses.
To establish S65T GFP retrovirus producers, GP+E86 ecotropic
packaging cells were cotransfected with MSCV/SRa-S65T and pMC1NeoPolyA
(Stratagene; La Jolla, CA), or with MSCV/CAG-S65T and pMC1NeoPolyA
using DOTAP reagent (Boehringer Mannheim; Mannheim, Germany) as
previously described [15]. After selection with 0.8 mg/ml G418, the
resistant packaging cell colonies were isolated and analyzed by FACS,
and the bright colonies were selected. GP+E86 cells were also
lipofected with MSCV/S65Tneo, and the bulk supernatant of
G418-resistant cells was used as the titration reference on NIH3T3
cells.
Retroviral Transduction of Hematopoietic
Cells
All the retroviral transduction experiments were performed in the
P2 facilities, according to the institutional recombinant DNA
biosafety guidelines. M1 cells were retrovirally transduced with
ecotropic virus supernatant, following a fibronectin-assisted
infection protocol [16]. One million M1 cells were incubated in 10
ml viral supernatant in a 100 mm Petri dish precoated with recombinant
human fibronectin fragment (RetroNectinTM, provided by
Takara Shuzo; Otsu, Japan). After an initial infection for 2 h, the
medium was replaced with fresh viral supernatant for an additional
transduction for 22 h. GFP fluorescence of the infected M1 cells was
analyzed by FACS at two days postinfection. According to the
fluorescence intensity, GFP-positive and -negative cells were
fractionated by FACS for further characterization.
Retroviral infection of murine BM cells and spleen colony assay
were performed using a standard coculture method [15]. C57BL/6 mice were injected
i.v. with 150 mg/kg 5-fluorouracil ([5-FU]; F. Hoffman-La Roche;
Basel, Switzerland), and BM cells were obtained two days
postinjection. The cells were plated at a density of 2 ¥
106 cells/ml and prestimulated for two days with 100 ng/ml
recombinant mouse stem cell factor (Genzyme; Cambridge, MA) and 100
U/ml recombinant human interleukin 6 ([IL-6]; provided by Ajinomoto;
Yokohama, Japan). On day 3, 4 ¥ 106 prestimulated cells
were plated on a 100-mm plate containing the viral producer cells
treated with mitomycin C (Kyowa Hakko; Tokyo, Japan), inoculated the
previous day at 3 ¥ 106 cells per dish and cocultured
for two additional days under the same growth factor conditions in the
presence of 4 µg/ml polybrene (Sigma; St. Louis, MO). In parallel,
the prestimulated BM cells were incubated on the untransfected GP+E86
packaging cells (mock infection). The infected cells were then
fractionated by FACS, according to the fluorescence intensity
generated by GFP. Lethally x-ray-irradiated (9 Gy) C57BL/6 recipient
mice were given 1 ¥ 104 FACS-fractionated cells per
animal i.v. to obtain individual colony-forming units spleen (CFU-S)
foci. Day 12 CFU-S (CFU-S12) foci were separated and
analyzed for GFP expression by
FACS.
|
Results
|
|---|
GFP Fluorescence of
Transfected M1 cells
M1 cells were transfected with several GFP constructs and assayed
for fluorescence intensity. By this assay, we evaluated the relative
brightness of WT and S65T GFP species and the efficiencies of CMV,
SRa, and CAG promoters. Figure
2 shows the fluorescence of the M1 transfectants after
cultivating at 37°C. WT GFP barely generated fluorescence when the
gene was driven by CMV or SRa promoter ( Fig. 2, panels A and B). CAG promoter induced
several-fold greater WT GFP expression, but considerable overlapping
of fluorescence was still observed between the transfected and the
untransfected cells (Fig. 2C). When S65T GFP was driven by CMV promoter, the
fluorescence was also minimal and hard to distinguish ( Fig. 2D). When driven by SRa or CAG
promoter, however, S65T GFP emitted green light—that is, M1
cells with SRa-driven S65T GFP were tenfold brighter than the
untransfected cells, while the transfectants with CAG-driven S65T GFP
emitted even more fluorescence (augmented by one- to two-log)
( Fig. 2, panels E and
F). These results indicated that S65T GFP was about tenfold brighter
than WT, and that CAG promoter showed the highest efficiency to
express GFP in M1 cells, followed by SRa.
View larger version (29K):
[in this window]
[in a new window]
|
Figure 2. FACS analysis of M1 cells transfected with WT and S65T GFP
expression constructs. Promoters and GFP species used for
transfection are CMV-WT (A), SRa-WT (B), CAG-WT (C), CMV-S65T (D),
SRa-S65T (E), and CAG-S65T (F). Dotted line, untransfected M1 cells;
bold line, transfected M1.
|
|
It has been reported that the WT and most of the derivative GFP
species have temperature sensitivity; i.e., the GFP proteins yield
greater fluorescence when synthesized at lower temperatures
[8]. We observed that the
incubation of the M1 transfectants at 30°C overnight prior to FACS
resulted in fluorescence augmentation of two- to fivefold, whether the
GFP species was WT or S65T (data not
shown).
Establishment of GFP Retrovirus
Producers
Based on the fact that S65T GFP showed tenfold brighter
fluorescence than WT in the transfected M1 cells, several retrovirus
vectors were constructed, as depicted in Figure 1. MSCV/SRa-S65T and MSCV/CAG-S65T have
efficient internal promoters but possess no drug-resistance
markers. These vectors were introduced into GP+E86 ecotropic packaging
cells, and isolated transfectants were analyzed by FACS for their
fluorescence intensities. The bright isolates were assayed for
supernatant infectivity on M1 cells, again by FACS ( Fig. 3B). An MSCV/SRa-S65T
transfectant yielded supernatant with the highest efficiency, and this
virus producer clone (EcoMSCV/SRa-S65T) was used in subsequent
transduction experiments. The RNA blot analysis of EcoMSCV/SRa-S65T
producers revealed that the molar ratio of the long terminal repeat
(LTR)-derived message (viral genomic RNA; 3.3 kb) and the SRa
promoter-derived mRNA (1.3 kb) was about 1:2 (data not shown). The
isolates of MSCV/CAG-S65T producers yielded supernatants with lower
titers than EcoMSCV/SRa-S65T (Discussion).
View larger version (22K):
[in this window]
[in a new window]
|
Figure
3. FACS analysis of S65T GFP retrovirus producers and
transduced M1 cells before and after sorting. (A)
EcoMSCV/SRa-S65T producer cells. Dotted line, untransfected GP+E86
packaging cells; thin line, EcoMSCV/SRa-S65T before sorting; bold
line, sorted EcoMSCV/SRa-S65T. (B) MSCV/SRa-S65T-transduced M1
cells. Dotted line, untransduced M1 control; thin line, bulk M1 cells
transduced by MSCV/SRa-S65T before sorting; bold line, sorted M1 cells
transduced by MSCV/SRa-S65T.
|
|
GP+E86 cells were also transfected with MSCV/S65Tneo vector, which
has the S65T GFP gene driven by the 5'-LTR promoter and a
PGK-neo cassette for G418 selection (Fig. 1). The ecotropic MSCV/S65Tneo supernatant was used
to standardize the viral titers on NIH3T3 cells by G418-resistant
colony-forming assay and RNA blot analysis. The RNA blot of NIH3T3
cells infected by MSCV/S65Tneo and MSCV/SRa-S65T was probed with a
radiolabeled S65T GFP cDNA for quantitation of the transgene messages
derived from the LTR. These signals were standardized with the
G418-resistant colony numbers resulting from MSCV/S65Tneo infection,
and the estimated titer of EcoMSCV/SRa-S65T supernatant on NIH3T3
cells was 5 ¥ 105 CFU per
ml.
Selection of Viral Producers and Transduced M1
Cells by FACS
During long-term passage, some EcoMSCV/SRa-S65T cells lost GFP
fluorescence and the virus titer decreased ( Fig. 3A, thin line). Selecting the brightest
producer population (top 2%) by FACS resumed the virus titer,
suggesting that the GFP expression in the packaging cells correlated
with viral production (Fig. 3A, bold line).
Fibronectin-assisted transduction of M1 with EcoMSCV/ SRa-S65T
supernatant typically yielded about 10% of the cells expressing GFP
( Fig. 3B, thin
line). These bright cells were readily fractionated by FACS as shown
in Figure 3B (bold
line). In another sorting, single bright cells were isolated and
expanded. All the recovered clones expressed GFP, with one- to two-log
greater fluorescence than the control M1 (not shown). These results
indicated that S65T GFP marker was useful for rapid selection of the
high-titer viral producers and the transduced
cells.
Transduction of Murine Bone Marrow and Spleen
Colony Assay
Based on the successful marking of M1 cells with S65T GFP, we next
transduced primary hematopoietic cells with MSCV/SRa-S65T vector. BM
cells obtained from 5-FU-treated mice were infected following a
standard coculture method. After two days of incubation on
EcoMSCV/SRa-S65T virus producers, BM cells were subjected to FACS. As
shown in Figure 4 , a
distinct cell population with green fluorescence was detected (panel
B, G3), which was not present in mock-infected bone marrow (panel
A).
View larger version (28K):
[in this window]
[in a new window]
|
Figure
4. FACS analysis of murine BM cells after MSCV/SRa-S65T
retrovirus infection.(A) Mock-infected BM. G1, collected
as "mock" fraction for CFU-S assay. (B)
MSCV/SRa-S65T-infected BM. G2, collected as
"GFP–" fraction for CFU-S assay; G3,
"GFP+" fraction for CFU-S
assay.
|
|
The cocultured BM cells were FACS-separated into three
populations, as illustrated in Figure 4. The first pool consisted of the cells incubated
on untransfected GP+E86 (mock; G1), the second population consisted of
the cells cocultured on EcoMSCV/SRa-S65T but without GFP fluorescence
(GFP–; G2), and the third was cultured on the viral
producers and had fluorescence (GFP+; G3). The frequency of
GFP+ BM cells was up to 5%, presumably because of
relatively low-titered viral producers (5 ¥ 105 CFU/ml;
Discussion). After sorting, the fractionated cells were injected
i.v. into lethally irradiated mice (1 ¥ 104 sorted
cells per animal) and assayed for CFU-S12. Each recipient
mouse had one to several spleen colonies on day 12, regardless of
which population of BM cells were given. This result suggested that
GFP expression in the transduced cells had no apparent disadvantage in
forming spleen colonies. Individual CFU-S12 foci were
isolated and evaluated for GFP expression by FACS ( Fig. 5). None of the spleen
colonies derived from the mock-infected or GFP– cells
had green fluorescence (Fig. 5, panels A and B), whereas most of the spleen
colonies derived from GFP+ cells had distinct fluorescence
( Fig. 5, panels
C-F). Table 1 summarizes
three independent CFU-S assays following BM transduction by
MSCV/SRa-S65T. Eleven of 12 (92%) spleen colonies derived from
GFP+-gated cells had green fluorescence, and fluorescent
spleen colonies were derived only from GFP+ donor
cells. These results clearly demonstrated that immature murine
hematopoietic stem/progenitor cells can be marked by MSCV/SRa-S65T
retrovirus and were readily fractionated. In addition, FACS analysis
of the individual spleen colonies allowed us to strictly evaluate our
separation efficacy; apparently the sorting windows chosen in this
study were stringent enough to discriminate transduced BM cells from
nontransduced cells. As shown in Figure 5, the constituent cells of each CFU-S12
focus derived from the GFP+-gated BM had markedly
homogeneous fluorescence (panels C-F). This result indicated that GFP
expression in transduced cells was maintained during proliferation and
differentiation at least until they formed spleen colonies, and
implies the feasibility of the GFP marker to track the progeny of the
gene-modified hematopoietic precursor
cells.
View larger version (29K):
[in this window]
[in a new window]
|
Figure 5. FACS analysis of representative spleen colonies.
Lethally irradiated recipient mice were i.v. injected with the
fractionated BM cells, and individual CFU-S12 foci
were analyzed by FACS. (A) A spleen colony derived from
"mock"-infected BM. (B) A spleen colony derived from
"GFP–" fraction of BM. (C-F)
Spleen colonies #1-4 derived from
"GFP+" fraction of
BM.
|
|
|
Discussion
|
|---|
In the present study, we showed that GFP is a suitable marker of
hematopoietic cells. Murine stem/progenitor cells were transducible
with a GFP-gene-containing retrovirus, and the successfully transduced
cells were readily separated. Others have recently shown transduction
of GFP genes into a variety of cells, including the hematopoietic
system [17-21]. Compared to a study by
Persons et al. [19], in which murine BM cells were transduced
more efficiently (about 40%), our transduction frequency was low (up
to 5%). The difference in transduction efficiency was presumably due
to the viral titer difference between these studies, i.e., Persons
et al. used high-titered retrovirus (5 ¥ 106
CFU/ml), whereas we used relatively low-titered virus (5 ¥
105 CFU/ml). In accordance with our results, Bagley et
al. reported that their retrovirus producers with similar titers
to our study (4-7 ¥ 105 CFU/ml) transduced small
fractions of murine BM (around 5%) [21]. Although S65T GFP had tenfold greater
fluorescence than the WT molecule, the unhumanized S65T GFP gene still
required very strong promoters such as SRa and CAG to generate
detectable fluorescence in hematopoietic cells. As has been
documented, strong internal promoters may interfere with transcription
from the proviral LTR and compromise viral titers, and incorporation
of introns into retroviral constructs may further complicate the titer
problem. Recent advances in engineering GFP genes allowed brighter
fluorescence and more efficient translation in mammalian cells
[18, 22]. We are currently evaluating
simpler retrovirus constructs containing humanized GFP genes, and some
of these vectors actually achieved higher titers (unpublished).
It is likely that expression of GFP selectable marker had little,
if any, detrimental effect on the in vivo proliferation and
differentiation of the hematopoietic progenitors to form spleen
colonies. FACS analysis of individual spleen colonies showed that
fluorescent CFU-S foci were exclusively derived from GFP+
BM cells, and the constituent cells of each colony displayed mostly
uniform fluorescence. This observation raises an intriguing question
of whether the GFP transgene will continue to express or shut down at
later stages of hematopoietic differentiation. In our preliminary
experiments with the simplified vectors, some peripheral blood cells
from the mice transplanted with GFP-transduced BM had fluorescence
(unpublished). Long-term and serial reconstitution studies will
provide further information about behavior of transgenes and
transduced hematopoietic cells in
vivo.
|
Conclusion
|
|---|
GFP selectable markers would greatly facilitate development of
hematopoietic stem cell gene therapy strategies by enabling rapid
selection of high-titer viral producers and genetically corrected
target cells and by providing systems for evaluating therapeutic gene
expression in vivo.
|
ACKNOWLEDGMENT
|
|---|
We are grateful to Drs. S. Takagi, R. Y. Tsien, K. Maruyama,
T. Naruse, R. G. Hawley, and A. Bank for research
materials and suggestions on GFP expression. We also thank Takara
Shuzo for RetroNectinTM, and Ajinomoto for IL-6. This
work was supported in part by Grants-in-Aid for Scientific Research
from the Ministry of Education, Science, Sports and Culture of
Japan.
|
References:
|
|---|
-
Dunbar CE, Cottler-Fox M, O'Shaughnessy JA et al. Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation. Blood
1995; 85
:3048
-3057.[Abstract/Free Full Text]
-
Kohn DB, Weinberg KI, Nolta JA et al. Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency. Nat Med
1995; 1
:1017
-1023.[Medline]
-
Sorrentino BP, Brandt SJ, Bodine D et al. Selection of drug-resistant bone marrow cells in vivo after retroviral transfer of human MDR1. Science
1992; 257
:99
-103.[Abstract/Free Full Text]
-
Phillips K, Gentry T, McCowage G et al. Cell-surface markers for assessing gene transfer into human hematopoietic cells. Nat Med
1996; 2
:1154
-1156.[Medline]
-
Pawliuk R, Kay R, Lansdorp P et al. Selection of retrovirally transduced hematopoietic cells using CD24 as a marker of gene transfer. Blood
1994; 84
:2868
-2877.[Abstract/Free Full Text]
-
Mavilio F, Ferrari G, Rossini S et al. Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer. Blood
1994; 83
:1988
-1997.[Abstract/Free Full Text]
-
Chalfie M, Tu Y, Euskirchen G et al. Green fluorescent protein as a marker for gene expression. Science
1994; 263
:802
-805.[Abstract/Free Full Text]
-
Cubitt AB, Heim R, Adams SR et al. Understanding, improving and using green fluorescent proteins. Trends Biochem Sci
1995; 20
:448
-455.[Medline]
-
Heim R, Cubitt AB, Tsien RY. Improved green fluorescence. Nature
1995; 373
:663
-664.[Medline]
-
Takebe Y, Seiki M, Fujisawa J et al. SRa promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol
1988; 8
:466
-472.[Abstract/Free Full Text]
-
Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene
1991; 108
:193
-200.[Medline]
-
Southern PJ, Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet
1982; 1
:327
-341.[Medline]
-
Hawley RG, Lieu FHL, Fong AZC et al. Versatile retroviral vectors for potential use in gene therapy. Gene Ther
1994; 1
:136
-138.[Medline]
-
Markowitz D, Goff S, Bank A. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol
1988; 62
:1120
-1124.[Abstract/Free Full Text]
-
Ding C, Kume A, Björgvinsdóttir H et al. High-level reconstitution of respiratory burst activity in a human X-linked chronic granulomatous disease (X-CGD) cell line and correction of murine X-CGD bone marrow cells by retroviral-mediated gene transfer of human gp91phox. Blood
1996; 88
:1834
-1840.[Abstract/Free Full Text]
-
Hanenberg H, Xiao XL, Dilloo D et al. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nat Med
1996; 2
:876
-882.[Medline]
-
Cheng L, Fu J, Tsukamoto A et al. Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells. Nat Biotechnol
1996; 14
:606
-609.[Medline]
-
Levy JP, Muldoon RR, Zolotukhin S et al. Retroviral transfer and expression of a humanized, red-shifted green fluorescent protein gene into human tumor cells. Nat Biotechnol
1996; 14
:610
-614.[Medline]
-
Persons DA, Allay JA, Allay ER et al. Retroviral-mediated transfer of the green fluorescent protein gene into murine hematopoietic cells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in vivo. Blood
1997; 90
:1777
-1786.[Abstract/Free Full Text]
-
Mazurier F, Moreau-Gaudry F, Maguer-Satta V et al. Rapid analysis and efficient selection of human transduced primitive hematopoietic cells using the humanized S65T green fluorescent protein. Gene Ther
1998; 5
:556
-562.[Medline]
-
Bagley J, Aboody-Guterman K, Breakefield X et al. Long-term expression of the gene encoding green fluorescent protein in murine hematopoietic cells using retroviral gene transfer. Transplantation
1998; 65
:1233
-1240.[Medline]
-
Cubitt AB, Woollenweber LA, Heim R. Understanding structure-function relationship in the Aequorea victoria green fluorescent protein. Methods Cell Biol
1999; 58
:19
-30.[Medline]
accepted for publication April 6, 1999.
This article has been cited by other articles:
|
|
|
|
 
J.-C. Tseng, B. Levin, T. Hirano, H. Yee, C. Pampeno, and D. Meruelo
In Vivo Antitumor Activity of Sindbis Viral Vectors
J Natl Cancer Inst,
December 4, 2002;
94(23):
1790 - 1802.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Arima, H. Toyama, H. Ichii, S. Kojima, S. Okada, M. Hatano, G. Cheng, M. Kubo, T. Fukuda, and T. Tokuhisa
A Putative Silencer Element in the IL-5 Gene Recognized by Bcl6
J. Immunol.,
July 15, 2002;
169(2):
829 - 836.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
