Opposing Roles of Syndecan-1 and Syndecan-2 in Polyethyleneimine-mediated Gene Delivery

Polyethyleneimines (PEIs) are efficient non-viral vectors for gene transfer. Heparan sulfate proteoglycans have been proposed to be the cell-surface receptors for PEI·DNA complexes (polyplexes). Here, we investigated if syndecan-1 (SDC1) and syndecan-2 (SDC2) are involved in PEI-mediated transfection. Following addition of polyplexes to HEK293 cells, green fluorescent protein-tagged SDCs rapidly formed clusters with PEI that were dependent of lipid raft integrity. However, although SDC1 overexpression slightly enhanced PEI-mediated gene expression, SDC2 dramatically inhibited it. Confocal microscopy analysis showed that SDC1·polyplex endocytosis occurred within minutes after addition of polyplexes, whereas SDC2·polyplex endocytosis took hours. Expression of SDC1 cytoplasmic deletion mutants revealed that the SDC1 cytoplasmic tail is required for gene expression, but not for clustering or endocytosis, whereas overexpression of SDC1/SDC2 chimeras showed that the SDC2 ectodomain is responsible for the inhibitory effect on gene transfer. This study provides evidence that SDCs may have opposing effects on PEI-mediated transfection. Polyethyleneimines (PEIs) are efficient non-viral vectors for gene transfer. Heparan sulfate proteoglycans have been proposed to be the cell-surface receptors for PEI·DNA complexes (polyplexes). Here, we investigated if syndecan-1 (SDC1) and syndecan-2 (SDC2) are involved in PEI-mediated transfection. Following addition of polyplexes to HEK293 cells, green fluorescent protein-tagged SDCs rapidly formed clusters with PEI that were dependent of lipid raft integrity. However, although SDC1 overexpression slightly enhanced PEI-mediated gene expression, SDC2 dramatically inhibited it. Confocal microscopy analysis showed that SDC1·polyplex endocytosis occurred within minutes after addition of polyplexes, whereas SDC2·polyplex endocytosis took hours. Expression of SDC1 cytoplasmic deletion mutants revealed that the SDC1 cytoplasmic tail is required for gene expression, but not for clustering or endocytosis, whereas overexpression of SDC1/SDC2 chimeras showed that the SDC2 ectodomain is responsible for the inhibitory effect on gene transfer. This study provides evidence that SDCs may have opposing effects on PEI-mediated transfection. Polyethyleneimines (PEIs) 2The abbreviations used are: PEIspolyethyleneiminesBPEIbranched 25-kDa PEIHSPGsheparan sulfate proteoglycansSDCssyndecansRITCrhodamine B isothiocyanateGFPgreen fluorescent proteinBFPblue fluorescent proteinDsRedDiscosoma sp. red fluorescent proteinhpthour(s) post-transfectionMβCDmethyl-β-cyclodextrin. 2The abbreviations used are: PEIspolyethyleneiminesBPEIbranched 25-kDa PEIHSPGsheparan sulfate proteoglycansSDCssyndecansRITCrhodamine B isothiocyanateGFPgreen fluorescent proteinBFPblue fluorescent proteinDsRedDiscosoma sp. red fluorescent proteinhpthour(s) post-transfectionMβCDmethyl-β-cyclodextrin. are cationic polymers that are synthesized as either linear or branched forms and are available over a wide range of molecular masses and polydispersities. Once added to DNA, PEIs associate with and condense DNA, forming PEI·DNA nanoparticles or complexes (termed polyplexes), protecting the DNA from nuclease degradation. The capacity of polyplexes to mediate gene transfer in mammalian cells was demonstrated for the first time by Boussif et al. (1Boussif O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Crossref PubMed Scopus (5621) Google Scholar). PEIs are currently attracting great interest in the field of gene therapy because they are relatively simple to synthesize, can be linked to a wide variety of molecules for cellular targeting, and may represent a promising and safe alternative to viral vectors (2Grzelinski M. Urban-Klein B. Martens T. Lamszus K. Bakowsky U. Hobel S. Czubayko F. Aigner A. Hum. Gene Ther. 2006; 17: 751-766Crossref PubMed Scopus (215) Google Scholar, 3Demeneix B. Behr J.P. Adv. Genet. 2005; 53: 217-230PubMed Google Scholar, 4Tang G.P. Guo H.Y. Alexis F. Wang X. Zeng S. Lim T.M. Ding J. Yang Y.Y. Wang S. J. Gene Med. 2006; 8: 736-744Crossref PubMed Scopus (170) Google Scholar). In addition, they offer an inexpensive and efficient vehicle for large-scale transfection applications, enabling the rapid production of recombinant proteins and viral vectors (5Pham P.L. Kamen A. Durocher Y. Mol. Biotechnol. 2006; 34: 225-237Crossref PubMed Scopus (175) Google Scholar). The linear and branched PEI (BPEI) 25-kDa forms have been shown to be among the most efficient non-viral vectors for in vitro (6Derouazi M. Girard P. Van Tilborgh F. Iglesias K. Muller N. Bertschinger M. Wurm F.M. Biotechnol. Bioeng. 2004; 87: 537-545Crossref PubMed Scopus (160) Google Scholar, 7Durocher Y. Perret S. Kamen A. Merten O-W. Mattanovich D. Lang C. Larsson G. Naubauer P. Porro D. Potsma P. Teixeira de Mattos J. Cole J. Recombinant Protein Production with Prokaryotic and Eukaryotic Cells: A Comparative View on Host Physiology. Kluwer Academic Publishers, Dordrecht, The Netherlands2001: 329-335Google Scholar, 8Boussif O. Zanta M.A. Behr J.P. Gene Ther. 1996; 3: 1074-1080PubMed Google Scholar) and in vivo (7Durocher Y. Perret S. Kamen A. Merten O-W. Mattanovich D. Lang C. Larsson G. Naubauer P. Porro D. Potsma P. Teixeira de Mattos J. Cole J. Recombinant Protein Production with Prokaryotic and Eukaryotic Cells: A Comparative View on Host Physiology. Kluwer Academic Publishers, Dordrecht, The Netherlands2001: 329-335Google Scholar, 8Boussif O. Zanta M.A. Behr J.P. Gene Ther. 1996; 3: 1074-1080PubMed Google Scholar, 9Goula D. Benoist C. Mantero S. Merlo G. Levi G. Demeneix B.A. Gene Ther. 1998; 5: 1291-1295Crossref PubMed Scopus (368) Google Scholar, 10Kircheis R. Schuller S. Brunner S. Ogris M. Heider K.H. Zauner W. Wagner E. J. Gene Med. 1999; 1: 111-120Crossref PubMed Google Scholar, 11Goula D. Remy J.S. Erbacher P. Wasowicz M. Levi G. Abdallah B. Demeneix B.A. Gene Ther. 1998; 5: 712-717Crossref PubMed Scopus (297) Google Scholar, 12Durocher Y. Perret S. Kamen A. Nucleic Acids Res. 2002; 30: E9Crossref PubMed Scopus (830) Google Scholar) gene delivery. A key characteristic of PEIs resides in their intrinsic buffering capacity, a feature also known as the proton sponge effect (1Boussif O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Crossref PubMed Scopus (5621) Google Scholar). According to one hypothesis, the high buffering capacity of PEIs leads to osmotic swelling and rupture of endosomes, resulting in the release of polyplexes into the cytoplasm and passive nuclear entry during mitosis when the nuclear membrane temporarily breaks down (13Grosse S. Thevenot G. Monsigny M. Fajac I. J. Gene Med. 2006; 8: 845-851Crossref PubMed Scopus (83) Google Scholar). polyethyleneimines branched 25-kDa PEI heparan sulfate proteoglycans syndecans rhodamine B isothiocyanate green fluorescent protein blue fluorescent protein Discosoma sp. red fluorescent protein hour(s) post-transfection methyl-β-cyclodextrin. polyethyleneimines branched 25-kDa PEI heparan sulfate proteoglycans syndecans rhodamine B isothiocyanate green fluorescent protein blue fluorescent protein Discosoma sp. red fluorescent protein hour(s) post-transfection methyl-β-cyclodextrin. Although it is generally accepted that membrane-associated heparan sulfate proteoglycans (HSPGs) play an important role in the uptake of polyplexes, their involvement as receptors for PEI·DNA polyplexes remains to be clearly demonstrated. The membrane-associated HSPGs encompass a wide variety of molecules, including betaglycan, also known as TGFR-3 (14Zhang L. Esko J.D. J. Biol. Chem. 1994; 269: 19295-19299Abstract Full Text PDF PubMed Google Scholar); CD44v3 (15Brown T.A. Bouchard T. St. John T. Wayner E. Carter W.G. J. Cell Biol. 1991; 113: 207-221Crossref PubMed Scopus (303) Google Scholar); syndecans (SDCs) (16Couchman J.R. Nat. Rev. Mol. Cell Biol. 2003; 4: 926-937Crossref PubMed Scopus (341) Google Scholar); and glypicans (17Lander A.D. Selleck S.B. J. Cell Biol. 2000; 148: 227-232Crossref PubMed Scopus (225) Google Scholar, 18Perrimon N. Bernfield M. Nature. 2000; 404: 725-728Crossref PubMed Scopus (659) Google Scholar). The SDC family is composed of closely related proteins (SDC1–4) encoded by four different genes. These proteins constitute the most abundant forms of membrane HSPGs and play a central role in several aspects of cell physiology (19Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2317) Google Scholar, 20Rapraeger A.C. Ott V.L. Curr. Opin. Cell Biol. 1998; 10: 620-628Crossref PubMed Scopus (101) Google Scholar, 21Carey D.J. Biochem. J. 1997; 327: 1-16Crossref PubMed Scopus (602) Google Scholar, 22Woods A. Couchman J.R. Trends Cell Biol. 1998; 8: 189-192Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) such as cell adhesion (23Woods A. Oh E.S. Couchman J.R. Matrix Biol. 1998; 17: 477-483Crossref PubMed Scopus (78) Google Scholar), modulation of growth factor activity (24Tkachenko E. Rhodes J.M. Simons M. Circ. Res. 2005; 96: 488-500Crossref PubMed Scopus (363) Google Scholar), and organization of the microfilament cytoskeleton (25Yoneda A. Couchman J.R. Matrix Biol. 2003; 22: 25-33Crossref PubMed Scopus (143) Google Scholar). The extracellular domain of SDCs, which show little primary sequence homology, possesses three to five glycosaminoglycan attachment sites and has a putative protease cleavage site in its extracellular juxtamembrane domain. SDCs have a highly homologous transmembrane domain, which is essential for their homodimerization and oligomerization (26Gao Y. Li M. Chen W. Simons M. J. Cell. Physiol. 2000; 184: 373-379Crossref PubMed Scopus (152) Google Scholar). The cytoplasmic domain possesses two highly homologous regions (C1 and C2) surrounding a variable region. The C2 region contains a PDZ-binding motif capable of interacting with numerous PDZ domain-containing proteins such as syntenin (27Grootjans J.J. Zimmermann P. Reekmans G. Smets A. Degeest G. Durr J. David G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13683-13688Crossref PubMed Scopus (345) Google Scholar), CASK/Lin-2 (28Cohen A.R. Woods D.F. Marfatia S.M. Walther Z. Chishti A.H. Anderson J.M. J. Cell Biol. 1998; 142: 129-138Crossref PubMed Scopus (321) Google Scholar), synbindin (29Ethell I.M. Hagihara K. Miura Y. Irie F. Yamaguchi Y. J. Cell Biol. 2000; 151: 53-68Crossref PubMed Scopus (104) Google Scholar), and synectin (26Gao Y. Li M. Chen W. Simons M. J. Cell. Physiol. 2000; 184: 373-379Crossref PubMed Scopus (152) Google Scholar). Notwithstanding their predominance as cell-surface HSPGs that display a high degree of structural and functional heterogeneity, no study has been undertaken to examine the potential role of syndecans in PEI-mediated transfection. In this study, we address the question of whether SDC1 and SDC2 as of PEI·DNA the of their overexpression on gene transfer and gene and their addition of that although SDC1 when in HEK293 cells, SDC2 it. The endocytosis, and of complexes the two SDCs, and this may be related to their transfection cytoplasmic domain deletion mutants of SDC1 and SDC1/SDC2 we the role of their in endocytosis and a of to PEI-mediated transfection and that SDC1 may as a for and were and and of The were and and rhodamine B isothiocyanate linear 25-kDa PEI and and DNA and DNA SDC1 and SDC1 deletion and SDC1/SDC2 chimeras were and in the mammalian C. J. B. Durocher Y. 2005; PubMed Scopus Google Scholar) with green fluorescent protein or blue fluorescent protein as in PEI was with by of a A. E. B. Mol. Ther. 3: Full Text Full Text PDF PubMed Scopus Google Scholar). was with in and for The was by the to a Cell and cells were in with and protein expression, cells were to the cells were PEI·DNA complexes as of microscopy and or microscopy was added to of DNA for and microscopy analysis or for been in of were and for their addition to the cell of and Discosoma sp. red fluorescent protein cells were by an with a cells were by to cells, and in a and of fluorescent proteins and was a were for cells with cells were with for the of were in for the cells with were with for were either on a a or on a The was with three A and a were used to the as an and a were used to the as and a and a were used to the as and were used for by microscopy and of SDC1 and SDC2 and that HEK293 cells or of or K. T. T. M. J. Biol. 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Full Text Full Text PDF PubMed Scopus Google Scholar), the important role of clustering for whether PEI can SDC1 and SDC2 polyplexes were added to or cells, and SDCs were by microscopy of addition, of clusters be for SDCs, but not for is as it was within after addition to A. and Y. most of the cell SDCs were in that of SDC1 and SDC2 proteins by polyplexes is of SDC1 and SDC2 was also addition of and linear 25-kDa PEI as as after addition of polyplexes with PEIs M. J.J. Chen J. Res. 2005; 22: PubMed Scopus Google Scholar). In no clustering be after addition of the cationic and was that clustering and endocytosis of SDC1 and the of membrane lipid Biochem. J. 2000; PubMed Scopus Google Scholar, E. E. Simons M. J. Cell Sci. 2004; PubMed Scopus Google Scholar). whether lipid raft is also required for clustering of and cells were for with a known to from the membrane and to lipid raft integrity. were added to cells, and SDC1 and SDC2 clustering was by and after and clustering of SDC1 and SDC2 was inhibited by Gene Expression by SDC1 but by the of SDC1 and SDC2 overexpression on PEI-mediated expression, cells or were with polyplexes a the of cells for SDC and and red for were by and to as PEI-mediated transfection of cells in an of in cells with transfection in cells were by with the SDC1 but SDC2 address this SDC1 and we the of polyplexes by microscopy PEI In with the SDC1 and SDC2 clustering be the cell after addition a and In addition, clusters with polyplexes, in with the capacity of SDC1 and SDC2 to PEI·DNA most SDC1 clusters were with polyplexes, in an to the and the In SDC2 clusters were the cell in with polyplexes several of polyplexes were of SDC1 clusters The polyplexes to be in as a of the putative of In a polyplexes were in cells and were with this In polyplexes of SDCs be in the cell cytoplasm a and In with a that entry of polyplexes into the during mitosis when the nuclear membrane is temporarily (13Grosse S. Thevenot G. Monsigny M. Fajac I. J. Gene Med. 2006; 8: 845-851Crossref PubMed Scopus (83) Google Scholar), polyplexes were also to the in cells whereas cells polyplexes in their after mitosis The of SDC1 for but for Gene gene was not but was slightly enhanced by overexpression of we to the of its with to PEI-mediated transfection. a of forms of SDC1 the C2 the variable and C2 the cytoplasmic and SDC1 ectodomain to the transmembrane which were in HEK293 microscopy analysis revealed that were the cell that not membrane were with polyplexes the and the of cells fluorescent proteins was by The are shown in and are to cells, as In cells, the of cells dramatically to a to that with SDC2 that the C2 domain is for PEI-mediated transfection. A in cells was for and Confocal microscopy analysis showed as after polyplexes to cells, PEI rapidly of clusters with forms of SDC1 for but were and with In to that the inhibitory effect of on cell transfection not be to endocytosis, but that the of polyplexes was These also that the C2 domain of SDC1 is for its gene The SDC2 but Gene to the role of the SDC1 and SDC2 during PEI-mediated gene delivery. address this we two chimeras and by the of SDC1 and the chimeras were with polyplexes the to their effect on transfection as by The show that of the SDC2 ectodomain with the SDC1 ectodomain transfection. In of the SDC1 ectodomain with the SDC2 ectodomain inhibited cell transfection to the as that the SDC2 ectodomain is responsible for the effect on cell transfection. Confocal microscopy analysis was with cells for SDC1 and clusters of chimeras with polyplexes be and in to endocytosis of complexes be However, although polyplexes were from as for they with for This that the ectodomain of SDC2 is responsible for the effect on endocytosis and gene SDC2 a on SDC1 and SDC2 have the capacity to polyplexes and clusters but dramatically in of endocytosis and transfection we the of their on SDC1 and SDC2 were with polyplexes the the of cells for SDC and red for was by The are to the SDC1 as of the was with to the of of SDC2 with SDC1 in an in transfection with of SDC1 that SDC2 has a effect on PEI transfection PEI-mediated transfection in cells was as a or of a endocytosis, microscopy analysis was after addition of polyplexes be to SDC1 and the of the SDC2 protein was with and polyplexes in clusters the complexes were within cells as they the cell as when SDC2 was This dramatically with the when SDC1 was that SDC2 has a effect on endocytosis and gene transfer to the high of recombinant protein expression, the non-viral gene vehicle the to and of cells, capacity to or the efficient to the and capacity to from DNA in the with which have to efficient PEIs display a relatively gene transfer The responsible for membrane and endocytosis of polyplexes In the of membrane for polyplexes and the of complexes to be clearly has been shown that HSPGs are involved in the cellular uptake of and cationic complexes J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). In the cell-surface HSPGs are syndecans and These molecules are involved in cellular such as and (19Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2317) Google Scholar). of are to extracellular proteins such as and their and growth and their In the to cell SDC1 overexpression has been to cationic transfection W. G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). However, no study to has the role of SDCs in PEI-mediated gene delivery. In addition, whether SDCs the capacity to and polyplexes in a that leads to efficient gene has not been In this we have demonstrated that this is not the as SDC1 and SDC2 have effects on PEI-mediated gene transfer in HEK293 HEK293 cells not to of SDC1 and the to which polyplexes and cells is not the of not to be in HEK293 cells, as a but in gene transfer was SDC1 of SDCs to be an important that their et al. V.L. G.P. J. 1997; PubMed Scopus Google Scholar) that of by SDC1 the of such SDC1 and we have clearly shown that addition of polyplexes rapidly and of clusters that These are with the of SDCs in PEI-mediated transfection and the first evidence that PEI·DNA complexes to and clustering of the no be or addition, that cationic transfection not SDC These to a study in which HSPGs and SDC1 were shown to cationic gene W. G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). However, as no evidence of SDC1 clustering was it is that HSPGs by of the cell In addition, it has been that the role of HSPGs in cationic transfection is to cells M. P. Biochem. J. 1999; PubMed Scopus Google Scholar). The of lipid has been shown to be essential for the clustering and activity of a wide variety of membrane proteins A. A. 2005; PubMed Scopus Google Scholar), including SDC1 and Biochem. J. 2000; PubMed Scopus Google Scholar, E. E. Simons M. J. Cell Sci. 2004; PubMed Scopus Google Scholar). we have shown that lipid raft is also required for clustering of SDC1 and that PEI-mediated gene is inhibited in a by in the of SDC1 and SDC2 on PEI-mediated gene Although by their it was that SDCs be membrane receptors for polyplexes, we that whereas SDC1 PEI-mediated gene expression, SDC2 it. Confocal microscopy analysis revealed that endocytosis of polyplexes to SDC1 occurred whereas that of polyplexes to SDC2 took hours. polyplexes be in the cytoplasm in that the of polyplexes in the cytoplasm is not to transfection of This is by that DNA remains in the for to in and cells E. S. Kamen Durocher Y. J. Biotechnol. PubMed Scopus Google Scholar). SDC1 was with its we the that polyplexes were with SDC1 although the inhibitory effect of SDC2 was not by SDC1 This that SDC2 has a effect on SDC1 and the in HEK293 Confocal microscopy analysis also revealed that SDC1 and SDC2 during PEI-mediated that their may The of endocytosis in cells as for with This in addition to endocytosis, the by which polyplexes cells may be an important for efficient gene transfer. the that the in endocytosis be responsible for the in cells, but is required to this The that SDC1 but not SDC2 is to the question SDC that are important for endocytosis, and gene a of SDC1 deletion we have shown that the C2 domain is for PEI-mediated gene expression, but not for clustering and the C2 domain can proteins a PDZ domain such as syntenin (27Grootjans J.J. Zimmermann P. Reekmans G. Smets A. Degeest G. Durr J. David G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13683-13688Crossref PubMed Scopus (345) Google Scholar), CASK/Lin-2 (28Cohen A.R. Woods D.F. Marfatia S.M. Walther Z. Chishti A.H. Anderson J.M. J. Cell Biol. 1998; 142: 129-138Crossref PubMed Scopus (321) Google Scholar), synbindin (29Ethell I.M. Hagihara K. Miura Y. Irie F. Yamaguchi Y. J. Cell Biol. 2000; 151: 53-68Crossref PubMed Scopus (104) Google Scholar), and synectin (26Gao Y. Li M. Chen W. Simons M. J. Cell. Physiol. 2000; 184: 373-379Crossref PubMed Scopus (152) Google Scholar), PDZ proteins may play a role in PEI-mediated transfection. Although to of proteins to be the of PDZ domain can also with M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is that the of the of SDC1 and SDC2 not with their with PDZ proteins such as In the of the SDC1 C2 domain is because its deletion PEI-mediated gene expression, but not clustering or This is in with a study that of the SDC PDZ-binding motif not SDC endocytosis P. Z. Degeest G. E. I. C. J. F. David G. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) In addition, the that polyplexes clustering of is in with a study that the SDC1 cytoplasmic domain is not required for its with lipid D.J. B. G. Cell Res. 1994; PubMed Scopus Google Scholar). endocytosis of was not which with that the SDC1 cytoplasmic domain is required for its with the cytoskeleton D.J. B. G. Cell Res. 1994; PubMed Scopus Google Scholar, D.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) and endocytosis Biochem. J. 2000; PubMed Scopus Google Scholar). is that polyplexes HEK293 with its This is as no be in cells, a in with that the SDC transmembrane domain is for SDC and oligomerization (26Gao Y. Li M. Chen W. Simons M. J. Cell. Physiol. 2000; 184: 373-379Crossref PubMed Scopus (152) Google Scholar). The of SDC1/SDC2 chimeras revealed that the ectodomain of SDC2 is responsible for the of PEI-mediated gene These that when to the SDC1 the cytoplasmic and transmembrane of SDC2 also have the capacity to endocytosis and gene This that SDC may represent an important to gene expression, on the that is in a of the SDC1 or for clustering and endocytosis were not whereas PEI-mediated gene was This clearly that endocytosis of polyplexes is not to gene expression, that or of polyplexes endocytosis is a for PEI-mediated gene transfer. This study provides into the involvement of in gene In we have shown for the first time that SDC1 and SDC2 are involved in and that SDC2 endocytosis and PEI-mediated gene this on the involved during transfection and also provides a for SDC1 and also that the role of of the family in PEI-mediated gene be investigated In addition, this that the of the SDCs in a or cell have a in gene therapy are to C. of and of for of SDC1 and SDC2 David for and of for the are to for with microscopy and to for with are to of for of the and D. and Zimmermann of for with

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