Unité de Recherche en Génétique Humaine, Centre Hospitalier de l'Université Laval, CHUQ, Faculté de Médecine, Université Laval, Sainte-Foy, Québec, G1V 4G2, Canada and 1 INSERM U 523, Institut de Myologie, Groupe Hospitalier Pitié-Salpêtrière, Paris, France
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Abstract |
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Keywords: cell transfection/Duchenne muscular dystrophy/EGFP/EGFP-dystrophin fusion proteins/electroporation/gene therapy/human dystrophin
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Introduction |
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Recently, green fluorescent protein (GFP) has been fused with many proteins at either the N- or C-terminal end, providing an interesting marker system for many research applications such as microscopy detection and flow cytometry (Cheng et al., 1996). To study the best delivery systems for mini-dystrophin and full-length dystrophin cDNAs, we have constructed fusion proteins consisting of `enhanced' GFP (EGFP) linked to the N-terminus of dystrophin and mini-dystrophin. In the present study, dystrophin expression cassettes were designed to be accommodated in viral or non-viral vectors in which the promoter can be removed and replaced by another without affecting the fusion protein. Also, the evaluation of the functionality of both constructs in vivo was further assessed by electroporation (Aihara and Miyazaki, 1998
) of dystrophin constructs in mouse skeletal muscles. Since dystrophin is mainly expressed as a subsarcolemmal protein in skeletal muscle fibers, we investigated whether the constructs could be expressed and would accumulate at relevant localizations in vivo in muscle tissue. In this paper, we report that the dystrophin fused to EGFP could be useful in a rapid and quantitative evaluation of the expression of recombinant dystrophin, both in vitro and in vivo.
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Materials and methods |
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Eukaryotic TA cloning kit containing the plasmid pCR3.1 expression vector was purchased from Invitrogen (Carlsbad, CA). EGFP cDNA was amplified by the polymerase chain reaction (PCR) of the gene contained in plasmid pIRES-EGFP (Clontech, Palo Alto, CA) using the primers C and D shown in Table I. Plasmid pRSVDMD.1 containing human full-length dystrophin cDNA was kindly provided by G.Karpati (Montreal Neurological Institute, McGill University, Canada). Dystrophin mini-gene (lacking exons 1748 of full-length dystrophin) was obtained by PCR amplification of DNA extracted from cells infected with an adenovirus containing the mini-dystrophin gene (Acsadi et al. 1996
; Moisset et al., 1998
). Primers A and B (Table I
) served for the amplification of mini-dystrophin cDNA with Expand High Fidelity DNA polymerase (Boehringer Mannheim, Montreal, QC, Canada) giving a final product of ~6 kb. The amplified mini-gene fragment was cloned directly in pCR3.1 vector (pCR3.1 mini-dystrophin). A series of six oligonucleotide primers were chemically synthesized using an ABI 394 synthesizer (Perkin-Elmer, Foster City, CA). These primers carrying specific restriction sites are listed in Table I
.
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We amplified with Taq DNA polymerase (Pharmacia Biotech, Montreal, QC, Canada) the EGFP cDNA (0.7 kb) from pIRES-EGFP using oligonucleotides C and D (Table I). The amplified product was cloned directly in pCR3.1 vector. Sense orientation of the amplified product (EGFP) in pCR3.1 was confirmed by restriction analysis and the construct was tested by transfection in Phoenix cells giving a fluorescent signal. The restriction sites NheI, KpnI and BamHI in the polylinker of the new construct were then deleted by cutting with restriction enzymes flanking these sites, blunting with Klenow enzyme (Pharmacia Biotech) and self-ligating with T4 DNA ligase (Pharmacia Biotech) resulting in pCR3.1 EGFP (Figure 1
).
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A DNA linker fragment (200 bp) joining EGFP to the 5' portion of the dystrophin cDNA containing the first BamHI site of dystrophin was obtained by PCR amplification of plasmid pRSV DMD.1 with primers E and F (Table I) and cloned in pCR3.1 vector (pCR3.1 link). This recombinant was sequenced (T7 sequencing kit, Pharmacia Biotech) for confirmation of the exact nucleotide sequence of the link between EGFP and dystrophin. The link DNA fragment was then obtained by a double digestion of pCR3.1-link with SalI and XhoI and cloned in the same sites found in pCR3.1 EGFP, making a recombinant pCR3.1 EGFP linked to the 5' portion of the dystrophin cDNA in which the stop codon carried by EGFP in pCR3.1 EGFP was eliminated (pCR3.1 EGFP5'Dys).
Generation of pCR3.1 EGFPmini-dystrophin (pMDysE)
We cloned in three steps the other DNA fragments obtained from the following digestions of pCR3.1 mini-dystrophin (pMDys) by directed cloning:
Generation of pCR3.1 EGFP full-length dystrophin (pDysE)
We cloned in two consecutive steps the other DNA fragments obtained by digestion of pRSVDMD.I and by digestion of pMDysE.
Transfection of EGFPdystrophin constructs into cultured cells
Phoenix cells lines (made by G.Nolan and provided by ATCC) were maintained in Dulbecco's modified Eagle's medium high glucose (DMEM; GIBCO/BRL, Burlington, ON, Canada) at 37°C under 5% CO2. Culture medium was supplemented with 10% fetal bovine serum (GIBCO/BRL), penicillin (10 000 IU/ml, GIBCO/BRL), streptomycin (10 µg/ml, GIBCO/BRL), sodium pyruvate (1 mM) (Sigma, St. Louis, MO) and glutamine (2 mM) (Sigma). Transfections of pMDysE and pDysE were performed with the Effectene transfection reagent (Qiagen, Mississauga, ON, Canada) according to the manufacturer's indications. The fluorescence signal was observed by microscopy after 24 and 48 h.
Protein extraction and Western blot analysis
For protein extraction, cells were rinsed twice with phosphate-buffered saline (PBS), lysed in 200 µl of 10 mM TrisHCl pH 7.5, 10% SDS, 1 mM dithiothreitol (DTT), 1 mM PMSF and treated by a procedure described by Wessel and Flügge (1984). Pellets resulting from the extraction procedure were partially dissolved in electrophoresis loading buffer (200 µl) containing 10% SDS and centrifuged before loading on the gel. Approximately 10 µg of protein (10 µl) were electrophoresed on 6% SDSPAGE gel and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 5% non-fat milk in PBSTween-20 (0.05%) (PBST). The primary antibody was NCL-Dys 2 (Novocastra Laboratories, Newcastle upon Tyne, UK), a monoclonal antibody directed against the carboxy-terminal 17 amino acids of dystrophin, diluted 1:300 in PBST with 1% non-fat milk. The secondary antibody was a goat anti-mouse antibody conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories) diluted 1:10 000 in PBST with 3% non-fat milk. The chemiluminescent signal was analyzed by a 2 min exposure to autoradiography film after detection with Renaissance reagent (NEN, Boston, MA).
Electroporation of mouse muscles with pMdysE or pDysE
Two-month-old C57BL6J mice were used for this study. Three experimental groups included three mice each and both Tibialis anterior muscles received the naked DNA and were electroporated. Each of the three groups received one of the following constructions: plasmid containing only EGFP, pMdysE and pDysE. Gene transfer was achieved using the ECM830 electroporator (Genetronics, San Diego, CA) according to the settings described by Mir et al. (1999). Briefly, 20 µl of naked DNA in solution in clinical-grade saline buffer (1 mg/ml) were injected into both Tibialis anterior muscles. An electric field was applied using plate electrodes. Given the potential immunogenicity of EGFP protein (Stripecke et al., 1999), animals were immunosuppressed with FK506 (2.5 mg/kg.day) starting on the day of electrotransfer (Kinoshita et al., 1994
).
Muscle examination
Five days after gene transfer, the animals were killed and the muscles were snap-frozen in isopentane cooled in liquid nitrogen. Serial cryostat sections were prepared throughout the whole muscle length, mounted in phosphate bufferglycerol (1:1) and observed under UV illumination (Zeiss microscope).
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Results and discussion |
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Previous reports have shown various levels of transfection in different cells in order to evaluate gene transfer of full-length or mini-dystrophin (Clemens et al., 1995; Acsadi et al., 1996
; Yanagihara et al., 1996
; McCaster et al., 1997
; Floyd et al., 1998
). However, a separate ß-galactosidase gene was usually used as a reporter gene in most cases. In the present study, transfection of pDysE or pMdysE constructs in Phoenix cells resulted in a good fluorescent signal in about 20% of the cells (Figure 2
). This observation was confirmed by Western blot analysis of equal amounts of Phoenix cell proteins (Figure 3
) showing bands of the expected size of 450 kDa for pDysE (Figure 3
, lane 2) and 240 kDa for pMDysE (Figure 3
, lane 3). Overall, these results show the suitability of EGFP fused to mini- or full-length dystrophin as a tool to determine rapidly the transfection efficiency in different cells instead of ß-galactosidase or EGFP alone.
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Notes |
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Acknowledgments |
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References |
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Aihara,H. and Miyazaki,J.-I. (1998) Nature Biotechnol., 16, 867870.[ISI][Medline]
Campeau,P., Chapdelaine,P., Moisset,P.A, Asselin,I., Venin,S., Massie,B. and Tremblay,J.P. (2000) Gene Therapy, submitted.
Cheng,L., Fu,J., Tsukamoto,A. and Hawley,R.G. (1996). Nature Biotechnol., 14, 606609.[ISI][Medline]
Clemens,P.R., Krause,T.L., Chan,S., Korb,K.E., Graham,F.L. and Caskey,C.T. (1995). Hum. Gene Ther., 6, 14771485.[ISI][Medline]
England,S.B., Nicholson,L.V., Jonhnson,M.A., Forrest,S.M., Love,D.R., Zubrzycka-Gaarn,E.E., Buman,D.E., Harris,J.B. and Davies,K.E. (1990) Nature, 343, 180182.[ISI][Medline]
Floyd,S.S. et al. (1998). Gene Ther., 5, 1930.[ISI][Medline]
Karpati,G., Gilbert,R., Petrof,B.J. and Nalbantoglu,J. (1997). Gene Ther., 10, 430435.
Kinoshita,I., Vilquin,J.-T., Guérette,B., Asselin,I., Roy,R. and Tremblay,J.P., (1994) Muscle Nerve, 17, 14071415.[ISI][Medline]
Koenig,M., Monaco,A.P. and Kunkel,L.M. (1988). Cell, 53, 219228.[ISI][Medline]
McCaster,G.C., Denetclaw JR,W.F., Reddy,P. and Steinhardt,R.A. (1997). Gene Ther., 4, 483487.[ISI][Medline]
McHowell,J.M. (1999). Neuromusc. Disord., 9, 102107.[ISI][Medline]
Mendell,J.R. et al. (1995) N. Engl. J. Med., 333, 832838.
Mir,L.M., Bureau,M., Gehl,J., Rangara,R., Rouy,D., Caillaud,J.M., Delaere,P., Branellec,D., Schwartz,B. and Sherman,D. (1999) Proc. Natl Acad. Sci. USA, 96, 42624267.
Moisset,P.-A., Gagnon,Y., Karpati,G. and Tremblay,J.P. (1998). Gene Ther., 5, 13401346.[ISI][Medline]
Partridge,T.A. and Davies,K.E. (1995) Br. Med. Bull., 51, 123137.[Abstract]
Stripecke,R., Carmen-Villacres,M., Skelton,D.C., Satake,N., Halane,S. and Kohn,D.B. (1999) Gene Ther., 6, 13051312.[ISI][Medline]
Wessel,D. and Flügge,U. (1984). Anal. Biochem., 138, 141143.[ISI][Medline]
Yanagihara,I., Inui, K, Dickson,G., Turner,G., Piper,T., Kaneda,Y. and Okada,S. (1996). Gene Ther., 3, 549553.[ISI][Medline]
Received March 25, 2000; revised July 1, 2000; accepted July 14, 2000.