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ÖZBAŞ, SUNA

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ÖZBAŞ

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SUNA

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2011 - 20173
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Has files: true × Subject: DNA ×

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Now showing 1 - 3 of 3
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    PublicationOpen Access
    Generation of stable cell line by using chitosan as gene delivery system
    (SPRINGER, 2016-08) EKENTOK ATICI, CEYDA; Salva, Emine; Turan, Suna Ozbas; Ekentok, Ceyda; Akbuga, Julide
    Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.
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    PublicationOpen Access
    Plasmid DNA-loaded chitosan/TPP nanoparticles for topical gene delivery
    (INFORMA HEALTHCARE, 2011-04) ÖZBAŞ, SUNA; Ozbas-Turan, Suna; Akbuga, Julide
    Topical application of plasmid DNA represents an attractive route of gene delivery. Although chitosan (CS) has been widely investigated as a gene-carrier, there is very limited information about the skin application of CS-based systems for DNA. This study evaluated pDNA-loaded chitosan nanoparticles (CS-NPs) for skin gene delivery. NPs were prepared by inducing the gelation of CS upon interaction with sodium tripolyphosphate. pSV-beta-Gal was used as a reporter gene. The size, surface charge, and the other in vitro characteristics of CS-NPs were examined. Primary human dermal fibroblast cells (HDF) and mouse fibroblast NIH 3T3 cell lines (ATCC CCL-92) were used for in vitro transfection studies. In in vivo study, CS-NPs were applied to the skin of baby and adult Sprague Dawley rats by spreading on the shaved area of the back of animals. During a week animals were sacrificed and skin biopsies were taken for beta-Gal expression. beta-galactosidase enzyme activity was determined spectrophotometrically at 420 nm. The distribution of beta-galactosidase expressing cells within the skin tissue was observed by X-gal histochemical method. beta-galactosidase was continuously expressed at the nanoparticle-treated skin during the 7 days. High and continuous beta-Gal expressions were obtained with CS-NPs, although it was low in the first day. When a comparison was made between the data of baby and adult rats, markedly high transfection were measured in the skin samples of the baby rats. NPs protected pDNA against the enzyme and serum attacks. In conclusion, CS-NPs showed in vivo transfection potential in rats for skin gene delivery.
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    PublicationOpen Access
    In vitro gene silencing effect of chitosan/shRNA PDGF-D nanoparticles in breast cancer
    (MARMARA UNIV, FAC PHARMACY, 2017-10-03) EKENTOK ATICI, CEYDA; Ekentok, Ceyda; Turan, Suna Ozbas; Akbuga, Julide
    Breast cancer is the most common cancer worldwide in women and it is highly malignant and fatal. PDGF-D plays role in regulation of many cellular processes such as angiogenesis. PDGF-D is overexpressed in many types of cancers and promote tumor growth and metastasis. Silencing of PDGF-D gene by using shRNA with an appropriate carrier system may decrease tumor growth and metastasis. In our study, we prepared chitosan nanoparticles loaded with five different shRNA plasmids targeting different exons of PDGF-D gene. Then, nanoparticles were characterized in vitro and transfection efficiency of these nanoparticles were investigated in breast cancer cell lines (MCF7, MDA-MB-231 and MDA-MB-435). The effects of single and multiple shRNA sequences, molecular weight of chitosan (150 kDa and 400 kDa) and the amount of shRNA (100 and 500 mu g) on the characterization and transfection efficiencies of nanoparticles have been studied. Size of nanoparticles changed between 200-400 nm and approximately 95-100% encapsulation efficiency were obtained. Release of shRNA changed with the molecular weight of chitosan. It was obtained that formulation containing shRNA plasmid targeting PDGF-D exon 6 (NP1) has the highest silencing efficiency in MDA-MB-231 cell line. It was also evaluated that chitosan can be a suitable gene delivery system for shRNA targeting PDGF-D.