Cationic Gelatin as a Gene Carrier

1995 ◽  
Vol 394 ◽  
Author(s):  
K. E. Brown ◽  
J. Bathon ◽  
C. H. Huang ◽  
R. Dalai ◽  
K. W. Leong
Keyword(s):  
Mammalian Cells ◽  
Viral Vector ◽  
Transfection Efficiency ◽  
Cell Types ◽  
Cell Transfection ◽  
Serum Free Medium ◽  
Free Medium ◽  
Mobility Shift ◽  
Dna Complexes ◽  
Dye Reduction

AbstractCationic gelatin was evaluated as a non-viral vector for cell transfection. We hypothesized that cationic gelatin would be a nontoxic alternative to already existing viral and non-viral cationic vectors. Cationic gelatin was synthesized by modifying gelatin with hexanediamine. Complexation of cationic gelatin with psv-β-gal plasmid caused an electrophoretic mobility shift of the plasmid. Cationic gelatin/DNA complexes were optimized in terms of transfection efficiency in CHODUK XB1 and COS 7 cell lines. Maximal gene expression for both cell types occurred in serum free medium with chloroquine (100 μM) at cationic gelatin/DNA ratios of approximately 2 and 7. In comparison with DEAE dextran, polylysine and Lipofectamine, cationic gelatin was the most efficient in transfecting COS 7 cells, with up to 18% cells transfected. In a dye reduction cytotoxicity assay, cationic gelatin caused < 5% of cells to become nonviable at a concentration of 100 μg/ml, while the other transfection reagents tested at the same concentration caused 25–100% of cell death. These results suggest that cationic gelatin holds promise as an effective vehicle for gene delivery to mammalian cells.

scholarly journals Synthesis, and Characterization, and Evaluation of Cellular Effects of the FOL-PEG-g-PEI-GAL Nanoparticles as a Potential Non-Viral Vector for Gene Delivery

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
S. Ghiamkazemi ◽  
A. Amanzadeh ◽  
R. Dinarvand ◽  
M. Rafiee-Tehrani ◽  
M. Amini
Keyword(s):  
Gene Delivery ◽  
Zeta Potential ◽  
Surface Charge ◽  
Cell Line ◽  
Graft Copolymer ◽  
Viral Vector ◽  
Transfection Efficiency ◽  
High Surface ◽  
Liver Cells ◽  
Dna Complexes

In this manuscript, we synthesized the potential non viral vector for gene delivery with proper transfection efficiency and low cytotoxicity. Polyethylenimine (PEI) is a well-known cationic polymer which has high positive surface charge for condensing plasmid DNA. However; it is highly cytotoxic in many cell lines because of the high surface charge, non-biodegradability and non-biocompatibility. To enhance PEI biodegradability, the graft copolymer “PEG-g-PEI” was synthesized. To target cancer liver cells, two targeting ligands folic acid and galactose (lactobionic acid) which are over expressed on human hepatocyte carcinoma were attached to graft copolymer and “FOL-PEG-g-PEI-GAL” copolymer was synthesized. Composition of this grafted copolymer was characterized using1H-NMR and FTIR spectra. The molecular weight and zeta potential of this copolymer was compared to PEI. The particle size and zeta potential of FOL-PEG-g-PEI-GAL/DNA complexes at various N/P ratio were measured using dynamic light scattering (DLS). Cytotoxicity of the copolymer was also studied in cultured HepG2 human hepatoblastoma cell line. The FOL-PEG-g-PEI-GAL/DNA complexes at various N/P ratios exhibited no cytotoxicity in HepG2 cell line compared to PEI 25K as a control. The novel copolymer showed enhanced biodegradability in physiological conditions in compared with PEI and targeted cultured HepG2 cells. More importantly, significant transfection efficiency was exhibited in cancer liver cells. Together, our results showed that “FOL-PEG-g-PEI-GAL” nanoparticals could be considered as a useful non-viral vector for targeted gene delivery.

The Influence of Physicochemical Parameters on the Efficacy of Non-Viral DNA Transfection Complexes: A Comparative Study

2006 ◽  
Vol 6 (9) ◽  
pp. 2776-2782 ◽  
Author(s):  
Carsten Kneuer ◽  
Carsten Ehrhardt ◽  
Heike Bakowsky ◽  
M. N. V. Ravi Kumar ◽  
Volker Oberle ◽  
...  
Keyword(s):  
Zeta Potential ◽  
Mammalian Cells ◽  
Transfection Efficiency ◽  
Dna Delivery ◽  
Dna Transfection ◽  
Dna Complexes ◽  
General Validity ◽  
Delivery Vehicles ◽  
Foreign Dna ◽  
Pronounced Reduction

Various polycationic vehicles have been developed to facilitate the transfer of foreign DNA into mammalian cells. Structure-activity studies suggested that biophysical properties, such as size, charge, and morphology of the resulting DNA complexes determine transfection efficiency within one class of vector. To investigate the general validity of these criteria, we studied the efficacy of a variety of DNA delivery vehicles including liposomes (DOTAP, SAINT2) with and without helper lipid (DOPE), the polymer polyethyleneimine (PEI), and cationic nanoparticles (Si26H, PLGA/chitosan) in a comparative manner. Sizes of the DNA complexes varied between 100 and 500 nm for PEI polyplexes and DOTAP/DOPE lipoplexes, respectively. The zeta potential was positive for PEI, Si26H, and DOTAP based complexes, while it was neutral for SAINT2-DNA complexes and negative for PLGA/chitosan-DNA complexes. The latter finding was elucidated by AFM, showing a layer of DNA adsorbed onto the nanoparticles. Transfection activity was negligible for PLGA/chitosan nanospheres, moderate for Si26H nanospheres and high for all other complexes, PEI being the most active carrier. The liposomal preparations were of low (DOTAP) or moderate (SAINT2) stability in serum, resulting in a pronounced reduction of gene expression, which was partially restored by the addition of chloroquine. In conclusion, transfection efficiency (i) seems to require a positive or neutral zeta potential, (ii) is depending on size, e.g., is higher for smaller particles, and (iii) requires a vector that is stable in serum.

High-Level Expression of Porcine Factor VIII from Murine Mesenchymal Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5281-5281
Author(s):  
Bagirath Gangadharan ◽  
H. Trent Spencer ◽  
Ernest T. Parker ◽  
Christopher B. Doering
Keyword(s):  
Gene Transfer ◽  
Ex Vivo ◽  
Cell Types ◽  
Mrna Levels ◽  
Serum Free Medium ◽  
Free Medium ◽  
High Level Expression ◽  
Serum Free ◽  
Fviii Activity ◽  
High Level

Abstract Low-level expression of human factor VIII (fVIII) has limited the success of clinical trials for hemophilia A using both ex vivo and in vivo gene transfer methods. Ex vivo genetic modification provides increased control of gene transfer and permits thorough characterization of transgene copy number, chromosomal integration site(s), and expression levels prior to transplantation. A subpopulation of adherent bone marrow-derived cell types, termed mesenchymal stem cells (MSCs), comprise an attractive target cell type for ex vivo gene transfer due to their accessibility, ability to differentiate into multiple cell types, and long-term survival following transplantation. Recently we demonstrated, in vitro, that recombinant B-domain-deleted porcine fVIII (rp-fVIII) is expressed at levels 10 – 14-fold greater than recombinant B-domain-deleted human fVIII (rh-fVIII) due to an enhanced rate of secretion from baby hamster kidney-derived (BHK-M) cells. Additionally we found that only the A1 and activation peptide-A3 domain sequences of porcine fVIII are necessary to retain high-level expression of hybrid human/porcine fVIII constructs. Here we report the expression of rp-fVIII from murine MSCs isolated from exon-16 fVIII knockout mice following ex vivo retroviral transduction. MSCs were transduced with ecotropic envelope-pseudotyped murine stem cell virus containing a rp-fVIIII transgene at an multiplicity of infection of 2 – 5 functional viral particles/target MSC. Following this transduction regimen the cell population harbored an average of 1.8 proviral genomes/cell as determined by quantitative real-time PCR. During culture in growth medium supplemented with 20% fetal bovine serum (FBS) rp-fVIII-transduced MSCs demonstrated a steady-state level of 2,400 fVIII mRNA transcripts/cell and an apparent fVIII production rate of 174 units/106 cells/24 hr as determined by quantitative real-time RT-PCR and one-stage clotting assay, respectively. However, when cultured in serum-free medium the mRNA levels decreased to 950 transcripts/cell and apparent fVIII production was reduced to 14 units/106 cells/24 hr. The latter mRNA/fVIII expression ratio is in agreement with that previously reported from stably transduced BHK-M clonal cell lines. In contrast the former mRNA levels are lower than predicted from the observed fVIII activity levels. The activation quotient (ratio of apparent fVIII activity following thrombin pre-treatment to non-thrombin treated baseline fVIII activity) increased 17-fold when the cells were cultured in serum-free versus serum-containing medium. Additionally, SDS-PAGE analysis of immunoprecipitated fVIII protein from serum-containing and serum-free medium revealed the presence of activated fVIII (fVIIIa) in conditioned medium samples containing FBS. Highly purified rp-fVIII from transduced-MSCs cultured in serum-free medium displayed similar relative mobility to BHK-M produced rp-fVIII upon SDS-PAGE analysis. These data warn against the determination of fVIII activity in serum-containing medium using the one-stage coagulation assay due to the presence of activated fVIII with high specific activity. Based on these results it is reasonable to predict that hemophilia A could be cured using high-level expression fVIII constructs by transplantation of a feasible number (106 - 108) of cells containing a single copy of rp-fVIII or other high-level expression hybrid human/porcine fVIII transgenes.

scholarly journals Human Thymic Epithelial Cells in Serum-Free Culture: Nature and Effects on Thymocyte Cell Lines

1992 ◽  
Vol 2 (2) ◽  
pp. 111-121 ◽  
Author(s):  
Carsten Ropke ◽  
Jette Elbroend
Keyword(s):  
Monoclonal Antibodies ◽  
Epithelial Cells ◽  
Cell Types ◽  
Thymic Epithelial Cells ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Factors ◽  
Serum Free ◽  
Functional Activities ◽  
Thymocyte Proliferation

Thymic epithelial cells (TEC) have been cultured for several months and/or for 4 to 5 transfers in a growth factor-defined serum-free medium without concurrent growth of other cell types. The use of monoclonal antibodies andαMAM-6 indicated that the majority of TEC were of medullary origin. The vast majority of cells were positive for LFA-3 and class I, and class II expression, was low or absent. Supernatants from the cultures were shown to contain IL-1ß, IL-6, and M-CSF. Coculture of cloned subpopulations of thymocytes and TEC showed effects of TEC and of secreted ILs on thymocyte proliferation. High percentages of TEC were able to bind DN, DP, or SP thymocyte populations, partly via CD2-LFA-3 adhesion. Thus, it is possible to culture TEC without unknown serum factors and with maintenance of functional activities.

CATIONIC POLYMER BASED GENE DELIVERY: UPTAKE AND INTRACELLULAR TRAFFICKING

COSMOS ◽  
2014 ◽  
Vol 10 (01) ◽  
pp. 17-24
Author(s):  
YOONKHEI HO ◽  
HENG-PHON TOO
Keyword(s):  
Gene Delivery ◽  
Transfection Efficiency ◽  
Cell Types ◽  
Endosomal Escape ◽  
Current Status ◽  
Uptake Mechanism ◽  
Nuclear Entry ◽  
Dna Complexes ◽  
Major Drawback ◽  
Gene Carriers

To date, low transfection efficiency remains the major drawback of polymer based gene delivery. Many cell types including stem cells, fibroblast and neurons are known to be poorly transfected with polymer based gene carriers and the high toxicity severely restrict their utility in gene delivery. Continual efforts are made to identify cellular barriers to efficient transfection as these carriers have low immunogenicity, ease of manufacturing and scalability. Here, we summarize the current status of understanding on uptake mechanism of polymer-DNA complexes (polyplexes), their endosomal escape, cytosolic transport and nuclear entry of pDNA.

scholarly journals Patterning of diverse mammalian cell types in serum free medium with photoablation

2009 ◽  
Vol 25 (2) ◽  
pp. 594-603 ◽  
Author(s):  
Vipra Dhir ◽  
Anupama Natarajan ◽  
Maria Stancescu ◽  
Anindarupa Chunder ◽  
Neelima Bhargava ◽  
...  
Keyword(s):  
Mammalian Cell ◽  
Cell Types ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free

Interferon production by mammalian cells grown in a serum-free medium

In Vitro ◽  
1979 ◽  
Vol 15 (5) ◽  
pp. 388-392 ◽  
Author(s):  
Robert W. Pumper ◽  
Leonard Molander
Keyword(s):  
Mammalian Cells ◽  
Interferon Production ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free

Trypsin sensitivity of mammalian cells grown in a serum-free medium

In Vitro ◽  
1971 ◽  
Vol 6 (4) ◽  
pp. 266-268 ◽  
Author(s):  
Robert W. Pumper ◽  
Peter Fagan ◽  
William G. Taylor
Keyword(s):  
Mammalian Cells ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free

Lipid metabolism in cultured cells. III. Cholesterol excretion process

1964 ◽  
Vol 207 (6) ◽  
pp. 1221-1225 ◽  
Author(s):  
J. Martyn Bailey
Keyword(s):  
Tissue Culture ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Rabbit Aorta ◽  
Rabbit Serum ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Cholesterol Excretion

Mammalian cells grown in tissue culture have been shown previously to take up considerable quantities of cholesterol from the growth medium. When cells grown on cholesterol-C14 supplemented medium were transferred to unlabeled medium containing serum, excretion of cholesterol into the outside medium took place. When cell cholesterol was labeled by intracellular synthesis from mevalonate-C14 precursor, it also was excreted readily into the serum medium. This excretion did not take place in serum-free medium and was found to be stimulated by a nondialyzable, thermolabile component of human serum. Horse, chicken, calf, and rabbit serum also showed stimulation ability. The process of cholesterol excretion appears to be of general occurrence. It was found in both strains of cultured cells examined (mouse fibroblasts and lymphoblasts) and also in strips of rabbit aorta incubated in vitro.

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