Development of a Self-assembling Nuclear Targeting Vector SystemBased on the Tetracycline Repressor Protein*

Received for publication, October 30, 2003Published, JBC Papers in Press, November 7, 2003, DOI 10.1074/jbc.M311894200

Laurence Vaysse‡, Richard Harbottle, Brian Bigger, Anna Bergau, Oleg Tolmachov,and Charles Coutelle§

From the Gene Therapy Research Group, Division of Biomedical Science, Imperial College London, Sir Alexander FlemingBuilding, Exhibition Road, London SW7 2AZ, United Kingdom

The ultimate destination for most gene therapy vec-tors is the nucleus and nuclear import of potentiallytherapeutic DNA is one of the major barriers for non-viral vectors. We have developed a novel approach ofattaching a nuclear localization sequence (NLS) peptideto DNA in a non-essential position, by generating a fu-sion between the tetracycline repressor protein TetRand the SV40-derived NLS peptide. The high affinity andspecificity of TetR for the short DNA sequence tetO wasused in these studies to bind the NLS to DNA as demon-strated by the reduced electrophoretic mobility of theTetR�tetO-DNA complexes. The protein TetR-NLS, butnot control protein TetR, specifically enhances gene ex-pression from lipofected tetO-containing DNA between4- and 16-fold. The specific enhancement is observed in avariety of cell types, including primary and growth-ar-rested cells. Intracellular trafficking studies demon-strate an increased accumulation of fluorescence la-beled DNA in the nucleus after TetR-NLS binding. Incomparison, binding studies using the similar fusion ofpeptide nucleic acid (PNA) with NLS peptide, demon-strate specific binding of PNA to plasmid DNA. How-ever, although we observed a 2–8.5-fold increase in plas-mid-mediated luciferase activity with bis-PNA-NLS,control bis-PNA without an NLS sequence gave a similarincrease, suggesting that the effect may not be becauseof a specific bis-PNA-NLS-mediated enhancement of nu-clear transfer of the plasmid. Overall, we found TetR-NLS-enhanced plasmid-mediated transgene expressionat a similar level to that by bis-PNA-NLS or bis-PNAalone but specific to nuclear uptake and significantlymore reliable and reproducible.

Nuclear translocation of a DNA-vector complex is a cruciallimiting step in non-viral transfection (1, 2). Several studieswith different transfection agents have shown that plasmid isefficiently internalized into cells but less than 1% of the DNApresent in the cytoplasm reached the nucleus (3, 4). In addition,several groups provided experimental evidence that cells un-dergoing mitosis are far more readily transfected than cell cyclearrested or quiescent cells, suggesting that the dissociation ofthe nuclear membrane during mitosis greatly facilitates nu-clear entry (5, 6).

The nuclear membrane is a tight barrier and transport oflarge macromolecules from the cytoplasm to the nucleus occursthrough a specialized structure of the nuclear envelope, thenuclear pore complex (for review see Refs. 7 and 8). Transportof proteins occurs by an energy-dependent process involvingthe interaction of specific highly basic nuclear localization se-quences (NLS)1 with the nuclear pore complex (9, 10). Thenuclear membrane is also a tight barrier for exogenous DNA orRNA and many proteins of the karyophilic viruses contain NLSsequences, which are involved in active transport of the viralgenome through the nuclear pore (11).

Several studies have shown that the addition of an NLSpeptide to non-viral gene transfer complexes can increase theirtransfection efficiency (12, 13). However, when the highly basicNLS peptide binds to DNA by electrostatic interaction, theincrease in nuclear import of the plasmid is modest (14, 15) andit seems that the direct interaction of the NLS peptide withDNA can impair its nuclear import capacity (16, 17). Chemicalmodification and random covalent coupling of peptides to agene expression cassette can also have detrimental effects (18,19). Little increase in nuclear targeting was obtained by use ofan NLS peptide linked to a DNA by triple helix formation witha single DNA target (20), whereas covalently linking an NLSpeptide to one end of capped linear DNA produced promisingresults (13). However, this technique is time consuming, expen-sive, and difficult to scale up.

To be efficient the NLS peptide should be able to bind withhigh affinity to a non-essential DNA sequence and remainaccessible for recognition by the nuclear pore complex. Here, wecompare two approaches for site-specific binding of an NLSpeptide to DNA. The first system exploits the tetracycline re-pressor protein TetR, a homodimer, with high affinity andspecificity for a palindromic short DNA sequence known as thetetracycline operator sequence tetO. In bacteria, in the absenceof tetracycline, the repressor binds to this sequence (associa-tion constant Ka 1011 M�1) inhibiting the expression of the twogenes it controls (21, 22). This system has also been widelyapplied to control gene activities in eukaryotic cells (23). In thisstudy, we have used its affinity for the operator sequence toattach an NLS peptide to plasmid vector DNA (Fig. 1A) byproducing a fusion protein between the TetR protein and thewell known NLS peptide from the SV40 large tumor antigen(13, 15) and have investigated the ability of the fusion proteinTetR-NLS to increase DNA nuclear import.

For comparison we have also exploited the ability of a di-meric homopyrimidine bis-PNA (peptide nucleic acid) (24, 25)to strand invade double stranded plasmid DNA and bind to a

* This work was supported by an F. D. Roosevelt grant from theMarch of Dimes of Birth Defects Foundation. The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

‡ Supported by Vaincre La Mucoviscidose.§ To whom correspondence should be addressed. Tel.: 44-2075943111;

Fax: 44-2075943015; E-mail: c.coutelle@imperial.ac.uk.

1 The abbreviations used are: NLS, nuclear localization sequence;X-gal, 5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside; MSC, mesen-chymal stem cells; PNA, peptide nucleic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 7, Issue of February 13, pp. 5555–5564, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 5555

by guest on Decem

ber 2, 2015http://w

ww

.jbc.org/D

ownloaded from

target site in a triplex interaction. Previously, binding of du-plex forming PNA-NLS to 11 target sites of plasmid DNA hasbeen used for nuclear import, resulting in enhanced DNA ex-pression by 5–8-fold (26), whereas binding of PNA-NLS to a15-bp fluorophore-tagged oligonucleotide was used to demon-strate nuclear entry (26, 27). This approach requires denatur-ation and reannealing of DNA plasmids for effective PNA bind-ing and has not resulted in widespread application, probablybecause of the technological demands of this cumbersomemethod. We hypothesized that the use of a bis-PNA binding bystrand invasion at room temperature to form a triplex struc-ture may enable us to use a bis-PNA-NLS conjugate for nucleartargeting using only a single target site in the plasmid DNA.

We demonstrate here a significant and reproducible enhance-ment of nuclear import and transgene expression by our TetR-NLS system in a variety of cell types, including primary andgrowth-arrested cells. By contrast, despite specific binding of allour PNAs to a single site in plasmid DNA, transfection enhance-ment is observed using either bis-PNA-NLS or bis-PNA alone atlow and high PNA:DNA binding ratios suggesting that this is notbecause of NLS-mediated nuclear localization.

MATERIALS AND METHODS

Construction of the TetR-NLS Fusion Protein and the Reporter GenePlasmid—Plasmids expressing the TetR-NLS protein or the TetR pro-tein were constructed using the pMal-c2 vector (New England Biolabs).To construct the pMal-TetRNLS plasmid, the tetR sequence was PCRamplified from pUHD15-1 using a primer encoding the NLS from theSV40 large tumor antigen (pkkkrkvedp) with an XbaI and EcoRI site atthe 5� and 3� end of the NLS. The amplification product was digestedwith EcoRI and inserted into the XmnI/EcoRI-digested pMal-c2 vectorusing blunt end and EcoRI ligation, resulting in the pMal-TetRNLSplasmid. The control plasmid pMal-TetR was obtained by digestion ofthe plasmid pMal-TetRNLS by XbaI and self-ligation to remove theSV40NLS sequence.

For construction of the reporter gene plasmids a XhoI/SacI fragmentcontaining seven tetO operator sequences (TCCCTATCAGTGATA-GAGA) was amplified by PCR from the pUHD10-3 plasmid using aprimer that inserts a BamHI site at the 5� end and a SalI site at the 3�end of the fragment. This BamHI/SalI fragment was then inserted intothe BamHI/SalI-digested pGL3-control plasmid (Promega). The finalconstruct pGL3-7tetO contains the luciferase reporter gene under con-trol of the SV40 promoter and seven TetR-binding sites (7tetO) in the 3�untranslated region of the expression cassette. Plasmid pGL3-2X7tetOcontaining the 7tetO fragment upstream and downstream from theexpression cassette was constructed by insertion of the 7tetO fragmentdigested with XhoI/SacI into the XhoI/SacI-digested pGL3 vector. Thesecond 7tetO fragment digested at the terminal BamHI and SalI siteswas then inserted into this BamHI/SalI-digested vector.

A linear DNA fragment of 2770 bp containing the reporter luciferasegene and the seven tet operator sequences at the 3� end (Frag7tetO) wasobtained by digestion of the plasmid pGL3-7tetO with BglII and SalI. Acontrol fragment containing only the reporter gene was obtained bydigestion with BglII and BamHI. A DNA fragment of 3052 bp(Frag2X7tetO) in which the luciferase gene is flanked by 7tetO se-quences 5� of the SV40 promoter and 3� to the untranslated region of theluciferase gene was obtained by digestion of plasmid pGL3–2X7tetOby SacI.

The fragment containing the 7tetO-binding site was also insertedinto a �-galactosidase expression plasmid using the SalI site. This8.7-kb construct, p�Gal-7tetO, contains the nuclear targeting �-galac-tosidase transgene under control of the cytomegalovirus promoter withthe 7tetO fragment at the 3� end.

Expression and Purification of the TetR-NLS Fusion Protein—E. coliDH5� carrying plasmids pMal-TetR or pMal-TetRNLS were grown at37 °C in LB medium containing 100 �g/ml ampicillin to A600 nm of 0.5.After addition of 100 �M isopropyl-1-thio-�-D-galactopyranoside to in-duce protein expression, the culture was incubated for 5 h. Cells wereharvested by centrifugation and the cell pellet was resuspended inprotein extraction buffer Bugbuster (Novagen). After an incubation of1 h at room temperature, cell lysates were cleared by centrifugation. Topurify the protein, the cell lysate was loaded onto an amylose resincolumn (New England Biolabs). Unbound proteins were removed byextensive washes with buffer containing 20 mM Tris-HCl (pH 7.4), 200

mM NaCl, 1 mM EDTA and the protein of interest was eluted in thesame buffer with 10 mM maltose. The protein-containing fractions werepooled, concentrated in an Amicon centricon unit and dialyzed over-night against 20 mM Tris-HCl (pH 8.0). The purified samples were thenanalyzed by SDS-polyacrylamide gel electrophoresis and quantified byBCA protein assay (Pierce), before storage at �20 °C. An average of 3mg of purified protein can be obtained per liter of bacteria culture.

Complex Formation and Binding Study of the TetR-NLS FusionProtein—The desired amount of DNA (plasmid or linear fragment),which contains the 7tetO fragment was incubated for 2 h at roomtemperature in 20 mM Tris-HCl (pH 8) containing 5 mM MgCl2 (21),with the fusion protein TetR-NLS or with the control protein TetR atdifferent molar ratios of dimer protein to plasmid. The same quantity ofprotein was used with the control DNA.

The protein�DNA complexes were loaded on a 1% agarose gel madeup in 1� TBE and containing 0.5 �g/ml ethidium bromide. For theinhibition binding experiment, the complex buffer was supplementedwith a tetracycline analogue, the doxycycline hydrochloride (Sigma).

PNA-NLS Molecules and Plasmid Constructs—J-bis-PNA H-(k)3TTJ-TTJTTTT(eg)3TTTTCTTCTTk-NH2 was the kind gift of Dr. Peter Nielsen(Center for Biomolekylaer Genkendelse, Copenhagen, Denmark). Controlbis-PNA H-(k)3TTCTTCTTTT(L)3TTTTCTTCTTk-NH2 and bis-PNA-NLS H-pkkkrkvedpyc(k)3TTCTTCTTTT(L)3TTTTCTTCTTk-NH2 weresynthesized by Oswel (Southampton, UK). Peptide nucleic acids are listedabove with PNA Watson-Crick bases capitalized and amino acids inlowercase. The lysines (k) added to bis-PNA-NLS and control bis-PNA aredesigned to improve PNA stability and hoogsteen binding to DNA becauseof their positive charge, and to give these PNAs a slight net positivecharge. L refers to a flexible AEEA linker (amino 2,6-dioxaoctanoic acid),whereas, eg refers to a similar flexible hydrophilic linker (8-amino 3,6dioxaoctanoic acid). J refers to the synthetic nucleobase pseudoisocy-tosine, which exhibits a positive charge. The SV40-modified NLS alone,H-pkkkrkvedpyc-NH2, was synthesized by Affiniti Research Products Ltd.(Mamhead, Exeter, UK).

Primers PNAXFwd 5�-TCGACTTTTCTTCTTG-3� and PNAXRev 5�-TCGACAAGAAGAAAAG-3� were used to insert a PNA-binding site, spe-cific for all of the PNAs, flanked by dual SalI sites in pDlox3 (28), makingpNOX3 (not shown). The CMV/luc cassette from pCIKLUX (28) wasexcised by BamHI and BglII digestion and inserted into the single BamHIsite of pNOX3 to create two constructs. The final constructs pNIX2 andpNIX10 contain the PNA-binding site at the end of the luciferase gene, orin front of the cytomegalovirus promoter, respectively.

PNA Binding and Assay for Restriction Digest Blocking—1 �l of PNAin 0.1% trifluoroacetic acid, with a final concentration of 0–20 �M, wasadded to 1 �l of template DNA (usually 40 or 200 ng). The reaction wasbuffered with 8 �l of TE at pH 8.0 or 9.0 as appropriate, to reach a pHof 7.6, in a 10-�l final volume of reaction buffer. This 10-�l reaction wasincubated at room temperature for 1 h, to achieve binding of PNA toDNA by strand invasion. The salt concentration was then adjusted to100 mM NaCl by the addition of 1.2 �l of 10� React 3 (1�, 50 mM

Tris-HCl, pH 8.0, 10 mM MgCl2, 100 mM NaCl) (Invitrogen) and 1 �l ofeither SalI (flanking the PNA-binding site) or EcoRI (80 bp downstreamof the PNA binding site), and incubated at 37 °C for 1 h. This reactionwas then run on a 1% agarose gel to visualize digestion of DNA.

Cell Transfection and Transgene Detection—NIH 3T3, HeLa, A549,COS7, and N18 cells were maintained in Dulbecco’s modified Eagle’smedium (Invitrogen) supplemented with 10% fetal calf serum. Primarymesenchymal stem cells (MSC) were derived from the femur of a neo-natal lamb. Briefly, whole marrow was flushed in phosphate-bufferedsaline and homogenized by fine chopping of the calcified material. A redblood cell lysis and filtration was performed and cells were washed inphosphate-buffered saline � 2% fetal calf serum before counting andresuspension in human mesencult medium (StemCell Technologies)according to the manufacturer’s instructions for human MSC deriva-tion. Sheep MSCs were derived by their ability to adhere to tissueculture dishes and their fibroblastic properties and cultured for a max-imum of two passages in human mesencult medium (StemCell Tech-nologies) prior to transfection.

Cells were seeded, 24 h before transfection, in 48- or 24-well tissueculture plates to reach 60 to 70% confluence during transfection. Thecomplexes TetR-NLS�DNA or PNA�DNA were prepared as describedabove with 0.25 or 0.4 �g of plasmid (depending on the tissue cultureplate) or the desired amount of linear fragment to give an identical genecopy number. The complexes were diluted in Opti-MEM (Invitrogen)and LipofectAMINE (Invitrogen) was added to the pre-formed DNAcomplexes at a DNA:liposome ratio of 1:12 (w/w) and incubated for 30min at room temperature to complete the lipoplex formation beforeadding to cells. After 3 h, transfection mixtures were replaced with

Self-assembling Nuclear Targeting Vectors5556

by guest on Decem

ber 2, 2015http://w

ww

.jbc.org/D

ownloaded from

complete medium. The transgene activity was measured after 24 husing a luciferase reporter assay kit (Roche Diagnostics) or a chemilu-minescent �-galactosidase reporter assay kit (Roche Diagnostics). Theprotein concentration of the cell lysates was determined using the BCAprotein assay (Pierce) and the enzyme activities were expressed asrelative light units per milligram of cellular protein. Each experimentwas performed at least three times.

For the in situ �-galactosidase visualization, cells were fixed withparaformaldehyde/glutaraldehyde solution and X-gal staining was per-formed. Cells were then counted, at least three separate times for eachwell to determine the transfection efficiency and each transfectionexperiment was repeated at least three times.

For transfection on cell cycle-arrested cells, cells were grown to60–70% confluence and the medium was replaced by Dulbecco’s modi-fied Eagle’s medium supplemented with 10% fetal calf serum andcontaining aphidicolin at 5 �g/ml (Sigma). After an incubation of 24 h,transfections were carried out as described above with medium contain-ing aphidicolin. For the experiment in the presence of doxycycline, thecomplex buffer and the cell medium were supplemented with doxycy-cline hydrochloride (Sigma) at a final concentration of 1 �g/ml.

Cell Trafficking Study—To label the fragment Frag2X7tetO, withoutdamaging the TetR-NLS binding recognition motifs tetO, the DNA wasdigested with MluI and SalI producing a 5� protruding end. The re-cessed 3� end of the fragment was then filled using exonuclease-freeKlenow-DNA polymerase (Amersham Biosciences) in the presence of 20�M unlabeled dATP, dCTP, dGTP, and Cy3-UTP for 1 h at 37 °C.Unincorporated nucleotides were removed using a QiaQuick purifica-tion column (Qiagen).

Cells were plated on glass coverslips for 1 day and transfections were

performed as described using 0.5 �g of labeled DNA. After 1 or 2 h at37 °C, cells were fixed with a solution of paraformaldehyde (3%) for 20min at room temperature. After several washes with phosphate-buff-ered saline, the slides were mounted in fluoro mounting medium (ICNBiomedicals) and observed on a fluorescence microscope.

RESULTS

Construction, Expression, and Purification of the Recombi-nant TetR-NLS Protein—A plasmid designed for bacterial ex-pression and purification of the protein TetR-NLS, which con-tains the TetR repressor in fusion with the SV40-derived NLS-peptide or the control protein TetR alone were constructed inthe pMal-c2 vector (New England Biolabs) (Fig. 1A). In thisvector, the cloned gene was inserted downstream from themalE gene of Escherichia coli, which encodes the maltose-binding protein, resulting in expression of TetR-NLS as anmaltose-binding fusion protein. After induction by isopropyl-1-thio-�-D-galactopyranoside the protein was overexpressed inbacteria and the fusion protein can be purified easily from thebacterial lysate on an amylose resin column using the affinityof the maltose-binding protein for maltose. As shown in Fig. 1Bthe resulting product can be found as a major band at 67 kDaon an SDS-polyacrylamide gel. Crucially, after dialysis in anappropriate buffer, the recombinant TetR proteins are able toform functional homodimers equivalent to the wild type TetRrepressor protein (29) (Fig. 1C).

FIG. 1. Expression and purification of recombinant TetR-NLS protein. A, principle of the self-assembling nuclear targeting vector systembased on the tetracycline repressor protein. B, SDS-PAGE analysis of the production and purification of the fusion protein TetR-NLS. Non-induced,lysate of E. coli cells non-induced by isopropyl-1-thio-�-D-galactopyranoside; Induced, lysate of E. coli cells induced by isopropyl-1-thio-�-D-galactopyranoside to overexpress the fusion protein; Flow through, cell lysate after loading on amylose column; Wash, extensive washes to removeunbound protein. Elutions 1–4, fractions of purified fusion protein eluted from amylose column with maltose. C, SDS-PAGE analysis of the purifiedfusion protein in native and denaturing conditions after concentration and dialysis against 20 mM Tris-HCl (pH 8).

Self-assembling Nuclear Targeting Vectors 5557

by guest on Decem

ber 2, 2015http://w

ww

.jbc.org/D

ownloaded from

TetR-NLS Protein Binds Specifically to tetO ContainingDNA—To investigate whether the TetR-NLS fusion proteincould specifically bind the tetO operator sequence as effectivelyas the TetR protein, we performed a gel retardation assay usingplasmid pGL3–7tetO carrying recognition motifs (tetO) at the 3�end of the luciferase gene (Fig. 2A). In accordance with theestablished uses of TetR/tetO interaction in gene expressionstudies (30) we constructed this vector with 7 TetR operatorsequences (7tetO). Both TetR and TetR-NLS proteins, respec-tively, appear as one major band of reduced electrophoreticmobility corresponding to protein-DNA complexes. A smallfraction of DNA was still able to migrate into the gel as non-complexed DNA. As already described for the wild type Tetrepressor (21), binding of the fusion protein is more efficient inthe presence of a low concentration of MgCl2. This bindingreaction is specific because gel retardation was not observedwith the control plasmid that does not contain the tetO se-quence. Similar experiments were performed with a linearDNA fragment containing the 7tetO sequence or with a controlfragment. Again retardation was achieved when the tetO se-quence was present but in the presence of MgCl2 no free DNA

was observed suggesting that binding of the protein to thelinear fragment is more efficient than to the circular plasmid.

As a further demonstration of specificity, we investigated theeffect of the antibiotic tetracycline on binding of the TetR-NLSto the tetO sequence fragment. The Tet repressor affinity forthe operator sequences is abolished by complexing it with theantibiotic (23, 31). In the TetR-NLS system, we found thattetracycline inhibits the binding only partially (data notshown) and therefore applied the more potent tetracycline an-alogue, doxycycline (23, 32). At a molar ratio Dox:TetR-NLS of1:1, the binding of the Tet repressor to the DNA was partiallyabolished. When the concentration of doxycycline was in-creased to a ratio 5:1, the DNA was completely released fromthe TetR-NLS protein and migrated freely into the gel (Fig.2B). This indicates that the formation of the TetR-NLS�DNAcomplex is indeed because of the binding between the TetR-NLS and the tetO sequence and not the result of nonspecificinteraction.

TetR-NLS Enhances Lipofection—To investigate the abilityof the TetR-NLS fusion protein to enhance gene transfer,protein�DNA complexes were formed at several different molar

FIG. 2. Gel retardation assay of fu-sion protein-DNA complexes. A, 1 �gof plasmid (plasmid pGL3–7tetO) or 3-kbfragment DNA carrying seven operatorsequences (7tetO) were incubated withthe fusion protein TetR-NLS, at a molardimer protein:DNA ratio of 5, withoutMgCl2 (TetR-NLS w/o MgCl2) or in thepresence of 5 mM MgCl2 (TetR-NLS). Theexperiment was performed in parallelwith DNA lacking the 7tetO sequence. B,fragment DNA carrying 7tetO was incu-bated with the fusion protein TetR-NLSin the presence of doxycycline (Dox) at amolar ratio for Dox:TetR-NLS of 1 or 5.Non-complexed DNA (DNA) or DNA com-plexed with the Tet repressor withoutNLS sequence (TetR) were used as a con-trol. The complexes were then electro-phoresed on a 1% agarose gel.

Self-assembling Nuclear Targeting Vectors5558

by guest on Decem

ber 2, 2015http://w

ww

.jbc.org/D

ownloaded from

ratios (1–10 corresponding to 10:250 ng to 100:250 ng) of pro-tein dimer to plasmid using the luciferase expression plasmidpGL3–7tetO and then adding the cationic lipid LipofectAMINEto the complexes. Because the addition of the protein can mod-ify the size of the lipid�DNA complexes and could thereby in-fluence transfection, the size profile of the different complexeswas studied by photon correlation spectroscopy. After 30 min,the size of the lipid�DNA complexes were 188 � 30 nm. A slightincrease in size was observed when the protein TetR-NLS orTetR was added to the complexes resulting in an average sizeof the complexes of 204 � 30 nm at a ratio of 5 and 216 � 30 nmat a ratio of 10.

The LipofectAMINE�protein�DNA complexes formed at dif-ferent ratios were used to transfect NIH 3T3 cells (Fig. 3). At amolar ratio of protein dimer/plasmid of 5:1, the fusion proteinTetR-NLS increases the transfection efficiency of the Lipo-fectAMINE more than 4-fold, whereas no enhancement oftransfection efficiency was observed with the control plasmidpGL3. These data indicate that the TetR-NLS protein enhancesgene transfer in a specific manner involving binding to the tetOoperator sequence and that this enhancement is because of thepresence of the NLS peptide as it was not observed with the Tetrepressor alone. Higher ratios of protein�DNA did not improvethe gene transfer.

FIG. 3. TetR-NLS fusion protein enhances lipofection. TetR-NLS�DNA complexes were prepared at a variety of protein dimer:DNA ratios usingeither plasmid pGL3–7tetO or pGL3–2X7tetO and the corresponding linear fragment of the plasmids containing 7tetO or two 7tetO and the expressioncassette (tet0 site). The corresponding DNA lacking the operator sequences was used as a control (w/o tet0). LipofectAMINE was then added to thecomplexes and transfections were performed on semiconfluent NIH 3T3 cells in 48-well plates. Luciferase activity was analyzed 24 h later and the levelsof enzyme activity per mg of protein were determined. For comparison, transfections were performed in parallel with the Tet repressor protein lackingthe NLS at a ratio of 5 or 20 (TetR) (hatched bars). The activity of the complexes were compared with the LipofectAMINE (Lipo), the numbers representthe luciferase activity (relative light units/mg of protein) of the different DNA construct with the LipofectAMINE alone.

Self-assembling Nuclear Targeting Vectors 5559

by guest on Decem

ber 2, 2015http://w

ww

.jbc.org/D

ownloaded from

Because the fusion protein binds the linear fragment moreefficiently and because we assume that efficient nuclear tar-geting of non-viral constructs also depends on a large extent onthe size of the transferred constructs, the same transfectionexperiments were done with a 2.7-kb DNA fragment containingonly the luciferase expression cassette with the 7 tetO sites atits 3� end (Frag7tetO). The amount of DNA used (125 ng) wascalculated to provide an identical number of transgenes as theintact plasmid (Fig. 3). The lipofection efficiency of the linearfragment was about 1 order of magnitude lower than plasmidlipofection. This is most likely because of degradation of theunprotected DNA ends by exonucleases. However, the enhance-ment of transfection by the TetR-NLS protein was significantlyhigher than with the DNA plasmid, about 8-fold of Lipo-fectAMINE alone. The highest increase was achieved with theFrag2X7tetO, which contains recognition motifs at both ends ofthe fragment. With this fragment, the TetR-NLS protein at aratio of TetR-NLS:DNA of 20 enhanced the transfection effi-ciency of the lipoplexes up to about 16-fold over LipofectAMINEonly. This increase is not just because of the protection of theDNA ends by the proteins because only a two times increase isobserved with the LipofectAMINE�TetR control. With the cor-responding plasmid, pGL3–2X7tetO, containing the 7tetO frag-ment both upstream and downstream from the expression cas-sette, the increase is around 5-fold that of LipofectAMINE, at aprotein dimer:plasmid ratio of 20:1. When the antibiotic doxy-cycline is added during transfection, the enhancement providedby the NLS fusion protein was ablated (Fig. 4), suggesting thatspecific binding of the TetNLS to the tetO recognition motifs isrequired for the enhancement of lipofection.

TetR-NLS Increases Nuclear Localization of TransfectedDNA—To study the intracellular localization of the complexes,we labeled the DNA Frag2X7tetO with the Cy-3 fluorophore.The label was applied at the end of the fragment in order not todestroy the binding site of the fusion protein. Two hours afterlipofection of NIH 3T3 cells with the DNA�TetR-NLS com-plexes, some localized fluorescence was observed by fluores-cence microscopy in the nuclei of many cells, whereas withLipofectAMINE alone the DNA was generally only observed inthe cytoplasm at this time point (Fig. 5). The proportion of cellscontaining DNA in the nuclei was evaluated by counting 100cells in two separate experiments. After 1 h lipofection, withthe TetR-NLS protein 35% of cells contained fluorescent DNA

in the nucleus in contrast to 11% cells with LipofectAMINEonly. After 2 h, these numbers reached to 51% with the TetR-NLS and 15% with the LipofectAMINE.

TetR-NLS Increases Transfection of Cell Cycle Arrested andPrimary Cells—To exclude cell division in the process of nu-clear entry, transfection experiments were also performed oncells pretreated with aphidicolin, which blocks cells in G1/Sphase (33) (Fig. 6). A dramatic decrease of the level of lipofec-tion with DNA only on these cell cycle-arrested cells is ob-served. However, addition of the fusion protein at the optimumdimer protein:plasmid ratio of 20 increases the transfectionefficiency 12-fold. An increase of 8-fold is also still obtainedwith the Frag2X7tetO. The enhancement of lipofection by TetR-NLS on arrested cells is a further indication of an NLS-medi-ated energy-dependent nuclear transfer of the TetR-NLS�DNAinvolving the nuclear pore complex.

To quantify the effect of TetR-NLS on transgene expressionin terms of the percentage of transgene expression as well as byoverall protein determination, we transfected several cell lines,including primary sheep MSC with a �-galactosidase plasmid.TetR-NLS enhanced �-galactosidase expression in all cell lines;about 3-fold on the easily transfected 3T3 cells and about 5-foldon the other cell lines. This activity increase is always associ-

FIG. 4. Inhibition of transfection by doxycycline. Lipofect-AMINE�TetR-NLS�DNA complexes were prepared at the optimum ratiousing the Frag2X7tetO as described in the legend to Fig. 3 in thepresence of doxycycline (DOX). Transfections were performed in NIH3T3 cells pretreated with doxycycline at a final concentration of 1 �g/ml.Luciferase activity was analyzed 24 h after transfection and expressedin relative light units (RLU)/mg of protein.

FIG. 5. Fluorescence microscopy with Cy3-labeled DNA. Thelinear fragment Frag2X7tetO labeled with Cy3 was used to transfectNIH 3T3 cells in combination with TetR-NLS�LipofectAMINE (top) orwith LipofectAMINE alone (bottom). Two hours after transfection, cellswere fixed and observed by fluorescent microscope (magnification �100).

Self-assembling Nuclear Targeting Vectors5560

by guest on Decem

ber 2, 2015http://w

ww

.jbc.org/D

ownloaded from

ated with an increase of the percentage of transfected cells.Interestingly, the best result was obtained in the primarysheep cell line, where the addition of the TetR-NLS enhancedthe percentage of transfected cells 3.5-fold (Fig. 7).

bis-PNAs Specifically Bind to a PNA Target Site in PlasmidDNA at Physiological pH—To compare our TetR-NLS systemwith another nuclear targeting system we tested the ability ofa PNA-NLS to increase DNA gene transfer. For this study, weused a bis-PNA displaying the SV40-derived NLS peptide (bis-PNA-NLS). The capacity of bis-PNA to strand invade doublestranded plasmid DNA and bind to a target site by triplexformation was exploited for binding of the NLS sequence to theDNA. J-bis-PNA (24) is known for its pH-independent triplexformation and clamping properties because of the presence ofpseudoisocytosine bases (J) on the second clamping strand.However, as we could not obtain a J-bis-PNA-NLS, we insteadconstructed bis-PNA as an exact homologue of J-bis-PNA ex-cept for the substitution of cytosine bases on the clampingstrand. The presence of pseudoisocytosine (J) is an importantcomponent of hoogsteen strand (triplex) binding kinetics (34),particularly at a physiological pH. Therefore, to further pro-tonate the hoogsteen strand of our bis-PNAs that lack Js weadded 3 lysine residues in the case of the control bis-PNA andthe positively charged NLS in addition for bis-PNA-NLS.

First, as for the TetRNLS protein, we investigated the abilityof the bis-PNA-NLS to specifically bind the target DNA se-quence, exploiting the ability of PNA to inhibit digestion ofDNA at an adjacent restriction site (35). We assayed for PNAbinding to DNA by blocking SalI restriction at sites flanking asingle PNA binding sequence in the plasmid pNIX2. In addi-tion, EcoRI restriction was used to demonstrate specificity. Asthe EcoRI site is distant from the PNA binding sequence, EcoRIdigestion should not be blocked by specific PNA binding (Fig.8). Incubation of the bis-PNA-NLS with the plasmid pNIX2 ata pH of about 7.6, to mimic a physiological environment, re-sulted in effective blocking of the two adjacent SalI restrictionsites as shown by the appearance of the different forms ofnondigested plasmid (supercoiled and opened circular). Thebinding of the bis-PNA-NLS is specific as EcoRI restriction ofpNIX2 was unaffected and no blocking of SalI restriction was

observed in the control plasmid pDLOX3 lacking PNA-bindingsite. There was a slight DNA retardation effect by bis-PNA-NLS resulting in an upwards band shift as seen in both SalI-and EcoRI-digested pNIX2 and to a lesser extent with thecontrol plasmid pDLox3 with increasing concentrations ofPNA. This is because of the positive charge present on bis-PNA-NLS and likely represents the enhanced binding effi-ciency of bis-PNA-NLS to pNIX2 as well as some nonspecificbinding to pDLox3.

No blockage of SalI digestion by specific PNA binding wasobserved at PNA:DNA molar ratios lower than 200:1 (data notshown). The presence of small amounts of linear DNA afterSalI digestion with pNIX2 indicates still incomplete binding ofbis-PNA-NLS at ratios of between 200:1 and 2000:1 (Fig. 8). Asimilar binding property was also observed with the controlbis-PNA that does not contain the NLS sequence, whereasbinding of J-bis-PNA resulted in almost complete restrictionenzyme blocking suggesting that binding was tighter and morespecific with this PNA (data not shown). At higher ratios ofbis-PNA-NLS:DNA, increased DNA gel retardation was ob-served indicating nonspecific binding by electrostatic interac-tions (data not shown). Taken together it can be inferred fromthese data that binding of our bis-PNA-NLS to DNA at ratiosbetween 200 and 2000:1 can be specific but is incomplete andrequires a large amount of bis-PNA-NLS.

Enhancement of Lipofection by bis-PNA-NLS—We then in-vestigated the effect of bis-PNA-NLS on the transfection effi-ciency of LipofectAMINE at several molar ratios of bis-PNA-NLS�DNA from 10:1 to 10,000:1 using a plasmid containing aPNA-binding site after the luciferase expression cassette (Fig.9). Addition of bis-PNA-NLS to DNA preparations at a low ratio(10:1) consistently produced a significant (p � 0.05) and repro-ducible 2–8.5-fold increase in luciferase activity over Lipo-fectAMINE alone on N18 and HeLa cells, respectively, whereasthis increase was not always reproducible on other cell lines.When higher ratios of bis-PNA-NLS to DNA were used (10,000:1), a highly significant (p � 0.01) 1.5–3 log increase in lucifer-ase activity was observed in all cells tested. However, theaddition of the control bis-PNA (without an NLS sequence) or ofthe control bis-PNA alone mixed with NLS peptide produced a

FIG. 6. Transfection experiment on cell cycle-arrested cells. LipofectAMINE�TetR-NLS�DNA complexes were prepared using the plasmidpGL3-7tetO or the Frag2X7tetO as described in the legend to Fig. 3. Transfections were performed in NIH 3T3 cells pretreated with aphidicolin(5 �g/ml) during 24 h. Luciferase activity was analyzed 24 h after transfection and expressed in relative light units (RLU)/mg of protein.

Self-assembling Nuclear Targeting Vectors 5561

by guest on Decem

ber 2, 2015http://w

ww

.jbc.org/D

ownloaded from

similar increase at ratios of 10:1 or 10,000:1, respectively.These data suggest that enhancement effects may not be be-cause of increased nuclear localization mediated by specificbinding of the NLS. As the larger effect occurred at bindingratios (10,000:1) causing retardation of DNA in agarose gels,this suggests that luciferase activity enhancement was mostlikely a result of nonspecific DNA condensation by bis-PNA-NLS or bis-PNA alone. Moreover, an almost identical enhance-ment of transgene expression was observed using these condi-tions with a control plasmid, which does not contain a PNA-binding site (data not shown). This emphasizes further that the

enhancement is not because of a specific bis-PNA-NLS bindinginteraction but most likely a result of nonspecific DNA conden-sation by PNA.

DISCUSSION

The aim of this study was to improve DNA nuclear translo-cation with non-viral vectors. It is often difficult in NLS studiesto establish the difference between an increase in gene expres-sion because of a genuine increase in active nuclear import ofDNA rather than as a result of DNA condensation by thecationic NLS sequence (17). The results of this study indicate

FIG. 7. Transfection with the TetR-NLS on different cell lines. LipofectAMINE�TetR-NLS�DNA complexes were prepared at differentratios using the plasmid p�Gal-7tetO. Transfections were performed on different cell lines including NIH 3T3, HeLa, A549 cells, and primary sheepMSC. A, �-galactosidase activity was analyzed 24 h after transfection and expressed in relative light units (RLU)/mg of protein. B, percentage oftransfected cells was determined after transfection with LipofectAMINE or TetR-NLS at the optimum ratio TetR-NLS:DNA of 5 by counting 100cells after X-gal staining. Cells were counted at least three separate times for each well to determine the transfection efficiency and eachtransfection experiment was repeated at least three times. C, typical fields after X-gal staining of MSC sheep primary cells transfected withLipofectAMINE alone or with LipofectAMINE�TetR-NLS�DNA complexes at a ratio TetR-NLS:DNA of 5:1.

FIG. 8. Specific blocking of restric-tion sites by binding of bis-PNA-NLS.Plasmids pDLox3 (3.4 kb) lacking, andpNIX2 (6.1 kb), containing a PNA-bindingsite, were incubated with increasingquantities of bis-PNA-NLS at PNA:DNAmolar ratios of 0, 200, 1000, or 2000:1 inTE buffer at pH 7.4 for 1 h. The buffer saltcontent was then increased and enzymedigestion was performed for 1 h with SalIor EcoRI. DNA samples were then electro-phoresed on an 1% agarose gel. The dif-ferent forms of the plasmid are indicated:open circular (oc), supercoiled band (ccc),and linear plasmid (linear).

Self-assembling Nuclear Targeting Vectors5562

by guest on Decem

ber 2, 2015http://w

ww

.jbc.org/D

ownloaded from

that the novel TetR-NLS fusion protein enhances lipofection inan NLS-specific manner by facilitating nuclear import. First,binding of the TetR-NLS fusion protein to DNA requires boththe presence of the TetR domain in the fusion protein and of thetetO recognition sequences in the DNA. The electrophoreticmobility shift assay demonstrated binding of the TetR-NLS tothe tetO recognition motifs without interference of the cationicNLS sequence. This binding is not because of electrostaticinteraction with the DNA via the NLS, because no gel retarda-tion of DNA without the tetO sequence is observed. Second,TetR-NLS binding can be completely abolished by doxycyclineas observed in the original TetR/tetO system (32). And finally,the 8–12-fold increase in gene expression also observed on cellcycle-arrested cells suggests that this increase is not dependenton cell mitosis but is an energy-dependent process involvingthe nuclear pore complex and the NLS sequence. The TetRprotein alone enhances the lipofection efficiency about twotimes, which may be because of the protection of DNA againstexonuclease activity notably for the linear DNA, and this in-crease is abolished by aphidicolin. In contrast, the TetR-NLSprotein increases the transfection efficiency of all DNA con-structs containing the tetO sequence.

Enhancement of transgene expression was shown here onseveral cell lines including primary mesenchymal stem cells.As these cells have stem cell properties (36) this may be im-portant for ex vivo gene therapy strategies. A comparison of theenhancement of gene transfer efficiency by determination oftotal transgenic protein and the percentage of transfected cellsshowed a less pronounced increase in percentage transduction.This is not surprising, as TetR-NLS would not increase the rateof cell entry of the construct but enhance nuclear transportonce entry has occurred.

The enhancement of transfection with the TetR-NLS proteinis dependent on the size or structure of the DNA construct. Forthe same copy number of transgenes, the increase is about 4times with the plasmid and about 16 times with theFrag2X7tetO on cell cycle active cells. The size of the DNAconstruct seems to be a crucial parameter in nuclear transfer

even with an active process. These data are in accordance withresults of other teams (37). The most successful study using theSV40 NLS peptide achieved an around 100-fold increase, usingshort DNA fragments (13), whereas only a 2–8-fold increasewas observed with plasmid (26, 38). It may be worth exploringdifferent pathways using our TetR-based system for the nu-clear import of larger DNA fragments. Increases of transfectionefficiency of plasmid up to 60-fold have been obtained using anon-classical NLS sequence from the heterogenous nuclear ri-bonucleoprotein, the peptide M9, which utilizes a differentnuclear protein import pathway involving transportin (39, 40).

Specific plasmid DNA binding could be achieved in our bis-PNA-NLS studies, refuting our initial concern regarding thebinding capacity of these bis-PNAs lacking pseudoisocytosinebases J. Specific digestion inhibition by bis-PNA-NLS con-structs at a ratio of 200:1, PNA:DNA, although somewhat re-duced compared with J containing PNA constructs, was clearlyeffective, suggesting continued PNA binding to DNA at physi-ological pH. This is in agreement with other demonstrations ofsuccessful binding of non-protonated bis-PNA at low pH (41), orJ-bis-PNA at high pH values (42), and suggests that we may bemost likely to obtain a dual PNA binding conformation at thespecific binding site on our plasmid (41), thus providing twoPNA-NLS peptides per plasmid molecule.

However, the specificity of bis-PNA-NLS enhancement ongene expression from a luciferase expressing plasmid was lessconvincing. A 2–8-fold enhancement of luciferase expressionwas observed by complexing of bis-PNA-NLS with DNA at alow ratio (10:1) that is considerably lower than that requiredfor observable PNA binding by restriction analysis. Althoughthis effect was variable between and within cell types, it wasoften statistically significant. However, control bis-PNA � NLSalone, added at this ratio to DNA, also produced similar levels ofluciferase enhancement (data not shown). The enhancing effectof PNA or NLS at this ratio cannot be because of condensation ofDNA at such low PNA concentrations as seen from our restrictioninhibition and retardation studies, suggesting an alternativemechanism of this enhancement of luciferase activity.

FIG. 9. Comparison of luciferase activity between 4 cell lines using different PNA constructs. Increasing concentrations of bis-PNA-NLS were bound to plasmid pNIX10, containing the luciferase expression cassette and a PNA-binding site, at ratios from 10:1 to 10,000:1PNA:DNA. Three controls were included at binding ratios of 10,000:1 of control bis-PNA, NLS alone, and control bis-PNA � NLS alone (10,000:1each), all bound at room temperature for 1 h at pH 7.6. LipofectAMINE was then added to the complexes and transfections were performed onsemiconfluent COS7, NIH 3T3, HeLa, N18 cells. Luciferase activity was analyzed 24 h after transfection and expressed in relative light units(RLU)/mg of protein.

Self-assembling Nuclear Targeting Vectors 5563

by guest on Decem

ber 2, 2015http://w

ww

.jbc.org/D

ownloaded from

The high concentration effect of PNA:DNA enhancement ofluciferase activity appeared to be solely because of charge/charge interactions leading to condensation of DNA. The en-hancing effect of oligolysine on cationic liposome-mediatedgene expression, by charge condensation of DNA (14, 43), sup-ports the hypothesis that the enhancement by high concentra-tions of PNA is because of the same effect. Interestingly NLSalone bound to DNA does not have an enhancing effect onluciferase activity at a 10,000:1 ratio. This is in agreement withdata on oligolysine enhancement of gene transfer, which showsthat oligolysines containing shorter lysine chains (�13) do notcondense DNA as well as, for example, Lys-18 (44, 45). Ourdata are in line with these observations because the NLSpossesses 3 overall positive charges, and bis-PNA-NLS has 7.However, the control bis-PNA, containing solely 4 positivecharges was also able to enhance luciferase activity at a10,000:1 ratio, suggesting a PNA specific enhancement effect,not related to specific PNA binding, nor solely to charge ratio.Indeed high molar ratio (10,000:1) control bis-PNA�DNA com-plexes transfected without LipofectAMINE into cells enhancedluciferase activity 5-fold compared with naked DNA, and werenot significantly less active than LipofectAMINE�DNA alone,suggesting that PNA has an effective condensation and possi-bly an uncharacterized protective effect for plasmid DNA incells (not shown).

The present study proves the principle and the utility of theTetR-NLS fusion protein in gene transfer with non-viral vec-tors. The TetR-NLS/tetO system presents several advantagesover PNA. The fusion protein can be expressed in bacteria andis easily purified from the cell lysate. The specificity of DNAbinding allows one to insert the recognition motif at differentnon-essential positions in the DNA, whereas DNA bindingoccurs spontaneously by mixing the DNA and fusion protein.Chemical modification reactions, which require relatively highamounts of purified material and sometimes result in productsthat are difficult to characterize are thus avoided. Furtherimprovement of the transfection efficiency of this system maybe achieved by optimizing the DNA construct using cappedDNA fragments (13) or minicircle DNA (28).

Our results support the use of multiple NLS sequences asobserved by Branden et al. (26), although the described DNAbinding techniques (strand invasion for PNA and hybridizationfor tet-NLS) are considerably more straightforward than an-nealing at high temperature as used by Branden et al. (26). Wealso surmise that had Branden et al. (26) used the control ofPNA uncoupled to NLS they may have observed increasedtransgene expression as we did, suggesting a still unexplainedexpression enhancing role for our control bis-PNA.

In conclusion we propose the use of the TetR-NLS system forex vivo and future in vivo applications in non-dividing cells.Similar levels of enhancement of transfection were also ob-served with TetR-NLS in combination with polyethyleneimine(data no shown) and as only a few fusion-protein molecules areadded per molecules of DNA, DNA�TetR-NLS complexes couldalso be used without a transfection agent for topical organinjection, e.g. into the muscle or liver.

REFERENCES

1. Uherek, C., and Wels, W. (2000) Adv. Drug Delivery Rev. 44, 153–1662. Nishikawa, M., and Huang, L. (2001) Hum. Gene Ther. 12, 861–8703. Pollard, H., Remy, J. S., Loussouarn, G., Demolombe, S., Behr, J. P., and

Escande, D. (1998) J. Biol. Chem. 273, 7507–75114. Labat-Moleur, F., Steffan, A. M., Brisson, C., Perron, H., Feugeas, O., Furst-

enberger, P., Oberling, F., Brambilla, E., and Behr, J. P. (1996) Gene Ther.3, 1010–1017

5. Zabner, J., Fasbender, A. J., Moninger, T., Poellinger, K. A., and Welsh, M. J.(1995) J. Biol. Chem. 270, 18997–19007

6. Wilke, M., Fortunati, E., van den Broek, M., Hoogeveen, A. T., and Scholte,B. J. (1996) Gene Ther. 3, 1133–1142

7. Gorlich, D. (1998) EMBO J. 17, 2721–27278. Macara, I. G. (2001) Microbiol. Mol. Biol. Rev. 65, 570–5949. Christophe, D., Christophe-Hobertus, C., and Pichon, B. (2000) Cell. Signal.

12, 337–34110. Jans, D. A., Xiao, C. Y., and Lam, M. H. (2000) Bioessays 22, 532–54411. Harel, A., and Forbes, D. J. (2001) Nat. Cell Biol. 3, E267–E26912. Collas, P., and Alestrom, P. (1996) Mol. Reprod. Dev. 45, 431–43813. Zanta, M. A., Belguise-Valladier, P., and Behr, J. P. (1999) Proc. Natl. Acad.

Sci. U. S. A. 96, 91–9614. Gao, X., and Huang, L. (1996) Biochemistry 35, 1027–103615. Fritz, J. D., Herweijer, H., Zhang, G., and Wolff, J. A. (1996) Hum. Gene Ther.

7, 1395–140416. Chan, C. K., Hubner, S., Hu, W., and Jans, D. A. (1998) Gene Ther. 5,

1204–121217. Cartier, R., and Reszka, R. (2002) Gene Ther. 9, 157–16718. Sebestyen, M. G., Ludtke, J. J., Bassik, M. C., Zhang, G., Budker, V.,

Lukhtanov, E. A., Hagstrom, J. E., and Wolff, J. A. (1998) Nat. Biotechnol.16, 80–85

19. Neves, C., Escriou, V., Byk, G., Scherman, D., and Wils, P. (1999) Cell Biol.Toxicol. 15, 193–202

20. Neves, C., Byk, G., Scherman, D., and Wils, P. (1999) FEBS Lett. 453, 41–4521. Hillen, W., Unger, B., and Klock, G. (1982) Nucleic Acids Res. 10, 6085–609722. Saenger, W., Orth, P., Kisker, C., Hillen, W., and Hinrichs, W. (2000) Angew.

Chem. Int. Ed. Engl. 39, 2042–205223. Baron, U., and Bujard, H. (2000) Methods Enzymol. 327, 401–42124. Larsen, H. J., and Nielsen, P. E. (1996) Nucleic Acids Res. 24, 458–46325. Nielsen, P. E., and Egholm, M. (1999) Curr. Issues Mol. Biol. 1, 89–10426. Branden, L. J., Mohamed, A. J., and Smith, C. I. (1999) Nat. Biotechnol. 17,

784–78727. Branden, L. J., Christensson, B., and Smith, C. I. (2001) Gene Ther. 8, 84–8728. Bigger, B. W., Tolmachov, O., Collombet, J. M., Fragkos, M., Palaszewski, I.,

and Coutelle, C. (2001) J. Biol. Chem. 276, 23018–2302729. Orth, P., Alings, C., Schnappinger, D., Saenger, W., and Hinrichs, W. (1998)

Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 99–10030. Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,

5547–555131. Hillen, W., Gatz, C., Altschmied, L., Schollmeier, K., and Meier, I. (1983) J.

Mol. Biol. 169, 707–72132. Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., and Bujard, H.

(1995) Science 268, 1766–176933. Escriou, V., Carriere, M., Bussone, F., Wils, P., and Scherman, D. (2001)

J. Gene Med. 3, 179–18734. Kuhn, H., Demidov, V. V., Nielsen, P. E., and Frank-Kamenetskii, M. D. (1999)

J. Mol. Biol. 286, 1337–134535. Nielsen, P. E., Egholm, M., Berg, R. H., and Buchardt, O. (1993) Nucleic Acids

Res. 21, 197–20036. Devine, S. M. (2002) J. Cell Biochem. Suppl. 38, 73–7937. Ludtke, J. J., Zhang, G., Sebestyen, M. G., and Wolff, J. A. (1999) J. Cell Sci.

112, 2033–204138. Chan, C. K., and Jans, D. A. (2001) Gene Ther. 8, 166–17139. Nakielny, S., Siomi, M. C., Siomi, H., Michael, W. M., Pollard, V., and Drey-

fuss, G. (1996) Exp. Cell Res. 229, 261–26640. Subramanian, A., Ranganathan, P., and Diamond, S. L. (1999) Nat. Biotech-

nol. 17, 873–87741. Hansen, G. I., Bentin, T., Larsen, H. J., and Nielsen, P. E. (2001) J. Mol. Biol.

307, 67–7442. Wittung, P., Nielsen, P., and Norden, B. (1997) Biochemistry 36, 7973–797943. Vitiello, L., Chonn, A., Wasserman, J. D., Duff, C., and Worton, R. G. (1996)

Gene Ther. 3, 396–40444. Wadhwa, M. S., Collard, W. T., Adami, R. C., McKenzie, D. L., and Rice, K. G.

(1997) Bioconjuguate Chem. 8, 81–8845. McKenzie, D. L., Collard, W. T., and Rice, K. G. (1999) J. Pept. Res. 54,

311–318

Self-assembling Nuclear Targeting Vectors5564

by guest on Decem

ber 2, 2015http://w

ww

.jbc.org/D

ownloaded from

Charles CoutelleBigger, Anna Bergau, Oleg Tolmachov and Laurence Vaysse, Richard Harbottle, Brian  Tetracycline Repressor ProteinTargeting Vector System Based on the Development of a Self-assembling NuclearGenes: Structure and Regulation:

doi: 10.1074/jbc.M311894200 originally published online November 7, 20032004, 279:5555-5564.J. Biol. Chem. 

  10.1074/jbc.M311894200Access the most updated version of this article at doi:

  .JBC Affinity SitesFind articles, minireviews, Reflections and Classics on similar topics on the

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/279/7/5555.full.html#ref-list-1

This article cites 45 references, 12 of which can be accessed free at

by guest on Decem

ber 2, 2015http://w

ww

.jbc.org/D

ownloaded from