Gene Therapy (2000)7,1063–1066
©2000Macmillan Publishers Ltd All rights rerved 0969-7128/00$15./gt
VIRAL TRANSFER TECHNOLOGY BRIEF COMMUNICATION
Plat-E:an efficient and stable system for transient packaging of retrovirus
S Morita,T Kojima and T Kitamura
Department of Hematopoietic Factors,Institute of Medical Science,University of Tokyo,4-6-1Shirokanedai Minato-ku,Tokyo 108-8639,Japan
A potent retrovirus packaging cell line named Platinum-E (Plat-E)was generated bad on the 293T cell line.Plat-E is superior to existing packaging cell lines regarding efficiency,stability and safety.The novel packaging constructs utilized in establishment of Plat-E ensure high and stable expression of viral structural proteins.Conventional packaging con-structs made u of the promoter of MuLV-LTR for expression of viral structural genes gag-pol and env,while our packaging constructs utilized the EF1␣promoter,which is 100-fold more potent than the MuLV-LTR in 293T cells in combination with the Kozak’s connsus quence upstream of the initiation codon resulting in high expression Keywords:p
ackaging cell;retrovirus;ecotropic;EF1␣promoter
Retroviral vectors and packaging cells are important tools for gene transfer applications.Introduction of retroviral vectors containing the gene of interest into suitable pack-aging cells enables production of infectious retrovirus,and the particles can infect target cells and stably trans-mit the gene of interest into chromosomes.In conven-tional strategies,stable high producers of a retrovirus vector harboring a gene of interest were established by transducing the retrovirus construct into NIH3T3-bad packaging cells such as PA317,1and 2–3months were usually needed to acquire high producers.Pear et al 2developed a unique packaging system by which high titer retrovirus can be obtained in 3days by transient transfection.The expression of viral structural genes was driven by the MuLV LTR in Bosc23cells.For transient transfection,the combination of Bosc23cells and the pMX-neo vector 3produced 1–3×106/ml virus,assd bad on the number of neomycin resistant col-onies of the infected NIH3T3cells (data not shown).Since Bosc23cells carry the large T antigen,we attempted to increa titers of the retrovirus by introducing the SV40origin to the pMX vector for amplification of the vector.However,this proved unfeasible (data not shown),sug-gesting that the limiting factor was the expression level of the viral structural proteins in the packaging cells.Bosc23was obtained by cotransfection of the plasmid encoding gag-pol together with the plasmid encoding the hygromycin-resistant gene and the plasmid encoding env
Correspondence:T Kitamura
Received 3November 1999;accepted 3March 2000of virus structural proteins in Plat-E cells.To maintain the high titers of retrovirus under drug lection pressure,we inrted the IRES (internal ribosome entry site)quence between the gene encoding gag-pol or env,and the gene encoding a lectable marker in the packaging constructs.Plat-E cells can stably produce retrovirus with an average titer of 1×107/ml for at least 4months.In addition,as we ud only the coding quences of viral structural genes to avoid inclusion of unnecessary retrovirus quences in the packaging constructs,the probability of generating the repli-cation competent retrovirus (RCR)by recombination can virtually be ruled out.Gene Therapy (2000)7,1063–1066.
together with the plasmid encoding another lection gene GPT (guanine phosphoribosyl transfera),one after the other.Therefore,expression of lectable markers did not guarantee the expression of gag-pol or env genes,which may account for the instability of the cells in producing high-titer retrovirus.
A similar packaging cell line Phoenix-E 4has also been developed.In Phoenix-E cells,the plasmids encoding the gag-pol and env genes were cotransfected with lection markers,which did not warran
t the stable expression of the gag-pol and env genes in the lection drug,hygromy-cin and diphtheria toxin,respectively.There were veral improvements in the Phoenix-E cells when compared with Bosc23cells.First,the RSV and CMV promoters,which are much stronger than MuLV-LTR in 293T cells,were ud to express the gag-pol and env genes,respect-ively.Second,the internal ribosome entry site (IRES 5)quence was ud to express gag-pol and a cell surface marker CD8simultaneously which enables sorting of high expresrs of the gag-pol gene.However,one needs to sort cells from time to time to maintain the expression levels of the gag-pol gene.
To design a packaging cell line which can stably pro-duce retrovirus with high titer,we arched for a strong promoter to drive expression of viral structural proteins in 293T cells using the FACS-GAL assay.6Among ven promoters tested,the EF1␣and CMV pro-moters induced high expression of lacZ (Figure 1).The activities driven by the promoters were 100-fold higher than tho by LTR utilized in Bosc23cells,and even exceeded tho by SV40and SR ␣promoters,which enable amplification of vectors in 293T cells expressing
Plat-E:a system for transient packaging of retrovirus
london bridgeS Morita et al
1064 Gene
英国 留学
Therapy
Figure1Activities of various promoters in293T cells.The activities of
the ven promoters,SV40,SR␣,EF1␣,RSV,TK,MuLV LTR and CMV
were evaluated by expression of lacZ under the control of each promoter in
293T7cells by the FACS-GAL assay as described.6Briefly,cells(1×106)healthyeatinghabits
transfected with each promoter construct were suspended in50l of phos-
phate buffered saline(PBS),then incubated for5min at37°C.FDG
(fluorescein di--d-galactopyranoside;Molecular Probes,Eugene,OR,
USA)was dissolved in distilled water,warmed at37°C and50l of2
m m FDG solution was added to50l of cell suspension.After1min of
incubation at37°C,1ml of PBS was added followed by incubation on ice
for2h.To stop the reaction,20l of50m m PETG(phenylethyl--d-
thiogalactoside;Sigma,St Louis,MO,USA)was added,and the prep-
aration was placed on ice until being subjected to FACS analysis.
the SV40large T antigen.Becau we thought that the
promoters of houkeeping genes were more suitable
than the viral promoters for driving stable gene
expression in mammalian cells,we ud the EF1␣pro-
moter to express the viral structural proteins in293T cells
(Figure2).In addition,IRES was inrted between the
gag-pol or env gene and the lection marker in the pack-
aging constructs described here.Therefore,expression of
the lection marker is a direct reflection of gag-pol or env
expression in the same cells.
Packaging constructs pEnv-IRES-puro r and pGag-pol-
IRES-bs r,which were constructed as described above,
were quentially transfected into293T cells and50sub-
clones resistant to both puromycin and blasticidin were
isolated.Among50subclones,clone1named Platinum-
E(Plat-E)produced the retrovirus which had the highest
infection efficiency and was ud for further analysis.The
titer of the retrovirus was about1×107/ml when tested
on NIH3T3cells,using rially diluted virus super-
natants of Plat-E cells transfected with pMX-lacZ(data
not shown).We next compared early passages of Plat-E
cells with tho of Bosc23cells and Phoenix-E cells with
regards to long-term stability to produce high-titer retro-
virus by transient transfection(Figure3).Culture con-
ditions of the three packaging cell lines were as follows:
Bosc23cells were grown in DMEM with10%fetal bovine
rum containing the GPT lection reagents as indicated
by the manufacturer(Specialty Media,Lavallette,NJ,
USA).Phoenix-E cells were sorted by FACS for
expression of CD8and maintained in DMEM with
10%
Figure2Schematic diagrams of packaging constructs.The packaging
羡慕英文
constructs ud for development of Plat-E are shown.The fragment carry-
ing the lectable marker,the blasticidin resistant gene(bs r)or the puro-
mycin resistant gene(puro r),was obtained by PCR using a pair of oligo-
nucleotides(for bs r:5Ј-AAAACATTTAACATTTCTCAACAAG-3Ј,5Ј-
ACGCGTCGACTTAATTTCGGGTATATTTGAGTG-3Ј,for puro r:5Ј-
ACCGAGTACAAGCCCACG-3Ј,5Ј-ACGCAGATCTTCAGGCACCG
GGCTTG-3Ј),and were inrted in the NcoI and SalI site(for bs r),or in
the NcoI and BglII site(for puro r)of pMX-IRES-EGFP.8The fragments
containing the IRES quence and either of bs r and puro r were excid
from the vector by NotI and SalI for bs r,or NotI and BglII for puro r,
respectively.The viral structural genes,gag-pol and env were amplified
by PCR,using the MoMuLV genome as a template,and the oligonucleo-
tide primers were ud as follows.Each primer contains either the EcoRI
site or the NotI site(underlined)and the5Јprimers also contain a Kozak’s
connsus quence GCCGCCACC located upstream of the initiation
codon.gag-pol:5Ј-CGAATTCGCCGCCACCATGGGCCAGACTGTT
ACCACTCCCTTAA-3Ј;5Ј-TACGCGGCCGCTCTGAGCATCAGAA
GAA-3Ј;env:5Ј-cGAATTCGCCGCCACCATGGCGCGTTCAACGCT
CTCAAAA-3Ј;5Ј-TACGCGGCCGCTATGGCTCGTACTCTAT-3Ј.The
resulting PCR fragments were digested with the EcoRI and the NotI frag-
ment.Finally,the fragment containing the viral structural genes,and the
fragment containing the IRES quence and the lection marker were
inrted downstream of the EF1␣promoter in the pCHO vector,a deriva-
tive of pEF-BOS.9For construction of the pGag-pol-IRES-bs r,pCHO was
digested with BamHI,and converted to a blunt end by Klenow reaction,
and then ligated with SalI linker(Stratagene,La Jolla,CA,USA).The
EcoRI–NotI fragment of gag-pol,and the NotI–SalI fragment of IRES-bs r
were inrted into the EcoRI and the SalI site of pCHO by triple ligation.
hung
To construct pEnv-IRES-puro r,pCHO was digested with EcoRI and
BamHI,and the EcoRI–NotI fragment of env,and the NotI–SalI fragment
of IRES-puro r were inrted into the EcoRI and the BamHI sites of pCHO.
Packaging constructs pEnv-IRES-puro r and pGag-pol-IRES-bs r were
quentially transfected into293T cells using Fugene(Boehringer
Mannheim,Germany)according to the manufactuer’s recommendations.
One day after transfection with pEnv-IRES-puro r,293T cells were lected
in DMEM containing1g/ml puromycin.The lected cells were then
transfected with the pGag-pol-IRES-bs r vector,and subcloned in the pres-
ence of puromycin and blasticidin(10g/ml).The lected clones were
tested for their potential to produce retrovirus.EF1␣,EF1␣promoter;
IRES,internal ribosome entry site;bs r,blasticidin resistant gene;puro r,
puromycin resistant gene.
fetal bovine rum containing hygromycin(300g/ml)
and diphtheria toxin(1g/ml)for1week,then cells
were transferred to DMEM with10%fetal bovine rum
without hygromycin and diphtheria toxin.Plat-E cells
were always maintained in DMEM with10%fetal bovine
rum containing blasticidin(10g/ml)and puromycin
(1g/ml).On one hand,infection efficiency of retro-
virus produced from Bosc23was decread within3
months,and that of retrovirus produced from the Pho-
enix-E cells also decread in time(Figure3).On the
other hand,Plat-E produced retrovirus with an infec-
tion efficiency greater than75%with a titer of about
1×107/ml for at least4months under drug lection
pressure.
To compare the expression level of gag-pol and env
mRNA in Plat-E,Bosc23and Phoenix-E packaging cell
Plat-E:a system for transient packaging of retrovirus S Morita et al
1065morpheus
Figure3Long-term stability of Plat-E in producing high titer retro-
virus.The infection efficiencies of Ba/F3cells using retrovirus derived
from pMX-GFP produced by Plat-E,Bosc23and Phoenix E were exam-
ined at the indicated times.pMX-GFP was constructed as follows.The
GFP fragment was excid from the pEGFP-N1vector(Clontech,Palo
Alto,CA,USA)by EcoRI and NotI,and was inrted into the EcoRI–
NotI site of the pMX vector.3Transfection and infection were performed
as described10except that we ud Fugene(Boehringer Mannheim)instead
of LipofectAmine(Gibco-BRL,Rockville,MD,USA).
nanna
lines,Northern blot analysis was done using the cells cul-
tured for3weeks.The expression levels of gag-pol and
env mRNA was four-fold and10-fold higher,respect-
ively,in Plat-E cells than in the other packaging cell lines
interpersonal communication(Figure4a).The RT activity in the cell lysate was also
analyzed.Plat-E produced at least twice more RT activity
when compared with Bosc23and Phoenix-E cells(Figure
4b).In addition,the expression level of env protein was
much greater than that of Bosc23and Phoenix-E(Figure
4c)when evaluated by antibody staining raid against
the env gene product.
As the retroviral structural genes were encoded on the
two different plasmids,three recombination events are
necessary to generate the replication competent retro-
virus(RCR).In addition,the probability of recombi-
nation was minimized by using only the coding quence
of gag-pol and env genes isolated by PCR from MuLV gen-
ome in the packaging constructs.In fact,production of
RCR was tested by the XC plaque assay,13and no RCR
was detected from Plat-E cells after transfection of pMX-
GFP.As for a positive control,a supernatant of
MoMuLV-infected C3H2K cells(a gift from Dr Hoshino)
was ud after rial dilutions,and the viral titer of the
wild-type MoMuLV produced from C3H2K cells was
estimated as1×104/ml.
In conclusion,we report here a stable retrovirus pack-
aging cell line Plat-E which has veral advantages over
the existing packaging cell lines.First,the EF1␣promoter
in the packaging constructs,in combination with the
Kozak’s connsus quence,allows production of retro-
virus with a titer of1×107/ml.Second,a bicistronic
vector carrying the IRES quence was ud in the pack-
aging constructs to ensure stable expression of the viral
structural genes under the drug lection pressure,which
Gene
Therapy
Figure4Comparison of gag-pol and env expression in Bosc23,Phoenix-
E and Plat-E.(a)Northern hybridization of gag-pol and env.Expression
of gag-pol and env in Plat-E(3)was compared with that in Bosc23(1)
and Phoenix-E(2)by Northern hybridization.The probes ud were the
EcoRI–NotI fragment of pGag-pol-IRES-bs r and pEnv-IRES-puro r.(b)RT
assay of cell lysate of Bosc23,Phoenix-E and Plat-E.Cell lysate of293T
cells was ud as a negative control(1),1ng of HIV RT was mixed with
the cell lysate of293T cells(2),and was ud as a positive control.RT
activities derived from Bosc23(3),Phoenix-E(4),Plat-E(5)were meas-
ured as described.11(c)Expression of env in Bosc23,Phoenix E and Plat-
E cells.The expression of env was determined by cell surfacefluorescence
of Bosc23,Phoenix-E and Plat-E using the rat monoclonal antibody raid
imco
against the env proteins termed83A25.12Staining procedure was perfor-
med as described12and then subjected to FACS analysis.As a control,
the cells were stained only with the cond antibody(FITC-conjugated
goat anti-rat IgG as cond antibody).
Plat-E:a system for transient packaging of retrovirus
S Morita et al
1066
Gene Therapy
makes it possible to maintain the titer of retrovirus derived from the Plat-E cells by simply culturing
the cells in the prence of lection drugs.Finally,to lesn the possibility of generation of RCR,the minimum virus quences were ud in the packaging constructs.Thus,Plat-E cells can stably produce helper-free retrovirus at high titers for a long time.
Using retrovirus produced by Plat-E cells,we can efficiently transfer genes to many different cells including cells in primary culture such as T cells and mast cells (data not shown).Recently,it has been reported that by introducing the coding region of the polyomavirus early gene into the packaging cell lines,the titers of recombi-nant retrovirus produced by the cell lines were 10–100times higher than tho produced by the parent cell line.14Introduction of the polyomavirus early region into Plat-E may lead to more efficient production of retro-virus with high titer.Plat-E is an ecotropic packaging cell line and generation of its amphotropic counterpart,the Plat-A cell line should prove uful in human gene therapy.
Acknowledgements
We would like to thank Dr Hiroo Hoshino (Department of Hygiene and Virology,Gunma University School of Medicine)for his kind gift of MoMuLV-infected C3H2K cells,and Dr Leonard H Evans (Laboratory of Persistent Viral Dia,National Institute of Allergy and Infectious Dia)for anti-E
nv antibody,and Dr Kunitada Shimo-tohno (Department of Viral Oncology,Institute for Virus Rearch,Kyoto University)for his valuable discussions.We also thank Ms Mariko Ohara for her providing langu-age assistance.This work was supported in part by grant-in-aid from the Ministry of Education,Science,Sports,and Culture and the Ministry of Health and Welfare of
Japan.The Department of Hematopoietic Factors is supported by Chugai Pharmaceutical Company Ltd.
References6级考试流程
1Miller AD,Buttimore C.Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.Mol Cell Biol 1986;6:2983–2902.
2Pear WS,Nolan GP,Scott ML,Baltimore D.Production of high-titer helper-free retrovirus by transient transfection.Proc Natl Acad Sci USA 1993;90:8392–8396.
3Onishi M et al .Application of retrovirus-mediated expression cloning.Exp Hematol 1996;24:324–329.4www.stanford.edu/group/nolan/.
5Gattas IR,Sanesm JR,Major JE.The encephalomyocarditis virus internal ribosome entry site allows
efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryo.Mol Cell Biol 1991;11:5848–5859.
6Fiering SN et al .Improved FACS-Gal:flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs.Cytometry 1991;12:291–301.
7Dubridge RB,Tang P,Hsia HC.Analysis of mutation in human cells by using an Epstein–Barr virus shuttle system.Mol Cell Biol 1987;7:379–387.
8Nosaka T et al .STAT5as a molecular regulator of proliferation,differentiation,and apoptosis in hematopoietic cells.EMBO J 1999;18:4754–4765.
9Mizushima S,Nagata S.pEF-BOS,a powerful mammalian expression vector.Nucleic Acids Res 1990;18:5332.
10Kitamura T et al .Efficient screening of retroviral cDNA
expression libraries.Proc Natl Acad Sci USA 1995;92:9146–9150.11Mathias S et al .Rever transcripta encoded by a human trans-posable element.Science 1991;254:1808–1810.
12Evans LH et al .A neutralizable epitope common to the envelope
glycoproteins of ecotropic,polytropic,xenotropic,and ampho-tropic murine leukeia virus.J Virol 1990;64:6176–6183.
13Rowe WP,Pugh WE,Hartly JW.Plaque assay techniques for
murine leukemia virus.Virology 1970;42:1136–1139.
14Yoshimatsu T et al .Improvement of retroviral packaging cell
lines by introducing the polyomavirus early region.Hum Gene Ther 1998;9:161–172.
