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

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

Animals ; Dependovirus ; genetics ; immunology ; Gene Transfer Techniques ; instrumentation ; Genetic Therapy ; instrumentation ; Genetic Vectors ; genetics ; immunology ; Humans

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

Gene-enhanced engineering deals with the scientific and technologic endeavour to produce cultured cells or polymer matrices transduced with multiple gene vectors encoding cytokine cDNA by means of genetic engineering technique, to make transduced cells or gene activated matrices highly express according cytokine, and then to enhance certain abilities of the artificial tissue. Up to now, various genes encoding modulatory species of ribonucleic of proteins such as growth factors, receptors, and transcription factors have been used in the context of gene-enhanced tissue engineering and expressed within numerous tissues, including artificial blood vessels, bone, cartilage, skin and urinary system, etc. Many experiments in vitro or in vivo have begun to show good prospects and great potential application of the new approach. We believe great changes will take place in the research field of tissue engineering due to the induction the of genetic engineering, and the new approach will become a very promising and valuable tool for therapy.
Blood Vessel Prosthesis ; Bone Substitutes ; Gene Transfer Techniques ; Genetic Engineering ; methods ; Tissue Engineering

OBJECTIVESTo investigate the effect of ionizing radiation on liposome-mediated gene delivery and find out a way to improve gene transfection.

METHODSPrior to liposome transfection, HR-8348 cells were irradiated at doses of 0, 2, 4, 8 Gy selected according to the surviving fraction line of HR-8348 cells after different dosage of radiation. After 36 h of liposome transfection, green fluorocytes were counted. The transfection efficiency was figured out and compared with each other.

RESULTSThe transfection efficiency of liposome-mediated gene delivery was 21.32%, 62.17%, 68.00%, 77.78% at the dose of 0, 2, 4, 8 Gy respectively and the clinical dose (2 Gy) was as high as 62.17%. Combined radiation and liposome-mediated gene delivery achieved the approximate transfection efficiency of virus vector.

CONCLUSIONIonizing radiation can improve the transfection efficiency of liposome-mediated gene delivery markedly and it is expected to treat human malignancy with liposome-mediated gene delivery combined with radiation.

Dose-Response Relationship, Radiation ; Gene Transfer Techniques ; Genetic Therapy ; Humans ; Liposomes ; Rectal Neoplasms ; therapy ; Transfection