Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings. However, antibody-mediated immunity, which includes both natural and induced antibody responses, is a major challenge for the success of pig-to-primate xenotransplantation. Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation. In this study, we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1, 3-galactosyltransferase gene GGTA1, which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation. Meanwhile, human leukocyte antigen-G5 gene HLA-G5, which acts as an immunosuppressive factor, was co-transfected with TALEN into porcine fetal fibroblasts. The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer. As a result, we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein. Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent. Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets. Functionally, the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs. These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.
Animals ; Animals, Genetically Modified ; Gene Knockout Techniques ; HLA Antigens ; Humans ; Nuclear Transfer Techniques ; Swine ; Transplantation, Heterologous

Animal models, particularly pigs, have come to play an important role in translational biomedical research. There have been many pig models with genetically modifications via somatic cell nuclear transfer (SCNT). However, because most transgenic pigs have been produced by random integration to date, the necessity for more exact gene-mutated models using recombinase based conditional gene expression like mice has been raised. Currently, advanced genome-editing technologies enable us to generate specific gene-deleted and -inserted pig models. In the future, the development of pig models with gene editing technologies could be a valuable resource for biomedical research.
Animals ; Gene Expression ; Gene Transfer Techniques* ; Mice ; Models, Animal ; Recombinases ; Swine

Comparing to Chitosan, Chitosan-oligosaccharides have several special functions, such as water-soluble, antitumor activity, immunostimulating effects, and antimicrobial activity. The chitosan-oligosaccharide, the molecular weight of which was about 5000, was used as research model. According to the agarose gel electrophoresis and UV spectrophotometer it was proved that electrostatic interaction was playing a very important role in the formation process of chitosan-oligosaccharide/DNA complex. The potential of adsorbing DNA on chitosan-oligosaccharide was analyzed by gel electrophoresis and UV spectrophotometer, and it was indicated that chitosan-oligosaccharide can improve the storage and structure stability of DNA. To check its protection ability to DNA by DNase I digestive experiment, the result showed that chitosan-oligosaccharide could load with plasmid effectively and protect DNA from being digested by DNase I. It was proved that chitosan-oligosacchide was safe and effective for gene delivery and will have a very good future in the field of gene therapy.
Chitosan ; chemistry ; DNA ; chemistry ; Gene Transfer Techniques ; Genetic Vectors ; Nanoparticles ; chemistry ; Oligosaccharides ; chemistry

OBJECTIVETo develop a novel non-viral gene delivery vector based on PEI-beta-CyD as backbone modified with aspirin, and to identify its physicochemical characters.

METHODS1, 1-carbonyldiimidazole (CDI) was used to bind aspirin onto PEI-beta-CyD to form PEI-beta-CyD-ASP. (1)H-NMR, FT-IR, UV and XRD were used to confirm the polymer structure. The ability of condensation was demonstrated by gel retardation assay. MTT assay was used to test the cell viability in B16, Hela and A293 cell lines. Transfection efficiency of the polymer was tested in B16 cells.

RESULTThe structure of PEI-beta-CyD-ASP was confirmed by (1)H-NMR, FT-IR, UV and XRD, which efficiently condensed plasmid DNA at the N/P ratio of 4. The copolymer showed low cytotoxicity and high transfection efficiency in B16 cells.

CONCLUSIONThe synthesized aspirin-PEI-beta-CyD might be a potential gene delivery vector.

Aspirin ; chemistry ; Cell Line ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Humans ; Polyethyleneimine ; chemistry ; beta-Cyclodextrins ; chemistry

OBJECTIVETo develop a novel vector for gene delivery with low molecular weight polyethylenimine grafted to the natural polysaccharide and conjugated to folic acid (LNT-PEI-FA).

METHODSThe properties of LNT-PEI-FA were characterized by (1)H-NMR, FT-IR and TGA, respectively. The particle size of LNT-PEI-FA/DNA complex was measured. The DNA binding ability of LNT-PEI-FA was detected by gel electrophoresis retardation assay.

RESULTThe particle size of LNT-PEI-FA/DNA complex was about 200 nm. Gel electrophoresis showed that at N/P ratio of 1.8 (W/W) the polymer was able to completely condense DNA. In vitro experiments showed a high efficiency of gene transfection in A293 and B16 cell lines.

CONCLUSIONA novel non-viral vector LNT-PEI-FA was successfully synthesized and characterized, which may be applied in gene transfection research in the future.

Cell Line ; Folic Acid ; chemistry ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Humans ; Lentinan ; chemistry ; Polyethyleneimine ; chemistry

OBJECTIVETo develop a novel gene delivery vector with poly-aspartamide-glutamic acid and polyethylenimine as the backbone.

METHODSalpha, beta-poly-(N-2-hydroxypropyl)-D, L-aspartamide-glutamic acid (PHPAG) was synthesized and low molecular weight polyethylenimine (PEI 1.8 kDa) was grafted to form PHPAG-PEI 1800. Chemical and biological characterization of the polymer was identified.

RESULTThe polymer was confirmed by (1)H-NMR, and the molecular weight was about 1.2 x 10(4). The ability of DNA binding was showed by gel retardation assay at N/P ratio of 3. 5. MTT assay showed that the polymer was non toxic in COS-7 and A293 cell lines. In vitro test demonstrated that it had high transfection efficiency in B16 and Hela cell lines.

CONCLUSIONPHPAG-PEI 1800 was successfully synthesized,which might be a potential vector for gene delivery.

Cell Line ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Glutamic Acid ; chemistry ; Humans ; Peptides ; chemistry ; Polyethyleneimine ; chemistry

DNA ; administration & dosage ; Gene Transfer Techniques ; trends ; Genetic Therapy ; methods ; Genetic Vectors ; Humans

OBJECTIVETo evaluate the impact of surface modification on the DNA-binding ability of nano-hydroxyapatite (nHA).

METHODSChemical co-precipitation-hydrothermal synthesis was utilized to prepare the nHA particles, and polyethylenimine (PEI) was used for surface modification of the nHA. Transmission electron microscopic (TEM) observation and zeta potential detection of the nHA were carried out before and after surface modification. The abilities of the nanoparticles, at different pH values and different concentrations, for DNA-binding and DNA protection against nuclease digestion were assessed before and after surface modification by electrophoresis.

RESULTSTEM observation showed a short rod-like morphology of PEI-modified nHA with uniform particle size and good dispersion; the nHA without the modification tended to aggregate with poor dispersion. With a positive zeta potential, the PEI-modified nHA showed an obviously enhanced ability of DNA binding at different pH values and concentrations, with strong capacity to protect the DNA against Dnase I digestion. At the concentration of 250 µg/ml and a pH value of 7.0, the nHA-PEI showed an optimal efficiency of DNA-binding and DNA protection.

CONCLUSIONnHA with surface modification by PEI can serve as an effective vector for DNA binding and transfer.

Amino Acid Motifs ; DNA ; chemistry ; Durapatite ; chemistry ; Gene Transfer Techniques ; Genetic Vectors ; Nanoparticles ; chemistry ; Polyethyleneimine ; chemistry

The intracellular trafficking and subcellular distribution of exogenous gene is very important for gene delivery. A successful gene vehicle should overcome various barriers including endosomal membrane barriers to delivery gene to the target organelle. Traditional nonviral vehicle is unable to avoid endosomal pathway efficiently, so the efficiency of gene delivery is low and the application of gene drugs is limited. In order to achieve efficient nonviral gene delivery, a lot of researches based on endosomal escape have been carried out and some agents with the function of endsomal escape have been found. These agents facilitate the endsomal escape via various mechanisms, such as fusion into the lipid bilayer of endosomes, pore formation in the endosomal membrane, proton sponge effect and photochemical methods to rupture the endosomal membrane. In this review, various reported strategies for endsomal escape are described according to the escape mechanisms, and their applications in intracellular gene delivery are also discussed.
Cell Membrane ; metabolism ; Endosomes ; metabolism ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Humans

OBJECTIVETo develop an alternative method for assessment of gene delivery systems in vivo.

METHODSMouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase (Gluc) expression cassette. After implantation of these cells into recipient mice, the expression of Gluc was detected in whole blood or plasma collected.

RESULTSAs little as 10 muL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer. And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice.

CONCLUSIONSGluc may be useful as an in vivo reporter for gene therapy researches, and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.

Animals ; Arecaceae ; enzymology ; Cell Line ; Gene Transfer Techniques ; Genes, Reporter ; Humans ; Luciferases ; genetics ; Mice