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PubMed2002

Efficient transfection method for primary cells.

Transfection of primary cells and stem cells is a problem in the laboratory routine and further in tissue engineering and gene therapy. Most methods working effectively for cell lines in culture fail to transfect primary cells. Here we describe the use of the Nucleofector technology developed by amaxa biosystems. We were able to transfect primary human melanocytes, human coronary smooth muscle cells, human chondrocytes, and human mesenchymal stem cells with high efficiencies (28.9-45.3%). All primary cell types failed to be transfected satisfactorily by methods based on liposome-mediated transfection in our hands. The viability of the transfected cells varied between 11.2% and 75% in comparison to untreated cells. Only 200,000 cells per transfection sample were needed. In summary, this method presents an effective and fast mean for transfection of primary and stem cells demonstrated by four cell types which are only transfected with low efficiency by other methods.

Cell Survival↗
PubMed2003

Efficient proliferation and adipose differentiation of human adipose tissue-derived vascular stromal cells transfected with basic fibroblast growth factor gene.

Human vascular stromal (VS) cells obtained from mature adipose tissue were transfected with an adenovirus vector carrying the basic fibroblast growth factor (bFGF) gene. bFGF protein was observed in VS cell nuclei 24 h after transfection and in the cytoplasm and extracellular space 72 h after transfection. Naive VS cells were almost static in vitro and proliferated in a dose-dependent manner on stimulation with recombinant bFGF (rbFGF). However, bFGF-transfected VS cells proliferated spontaneously to the same extent as naive VS cells when stimulated with rbFGF at 100 ng/ml. The former cells started to proliferate on day 3 after transfection and the proliferation pattern was similar to that of the latter cells, although only a slight amount of bFGF protein was detected in the culture medium when the bFGF-transfected cells started to proliferate. The proliferation of bFGF-transfected VS cells was completely inhibited by bFGF neutralizing antibody, which also completely inhibited the proliferation of naive VS cells stimulated with rbFGF. Under conditions favoring differentiation to adipocytes, bFGF-transfected VS cells stopped proliferating and started to accumulate lipid in the cytoplasm. bFGF-transfected VS cells, which spontaneously and efficiently proliferate while preserving their ability to differentiate into adipocytes, may be an adequate cell source for human adipose tissue regeneration.

Adipose Tissue↗
PubMed1994

Efficient transfection of primary cells in a canine hemophilia B model using adenovirus-polylysine-DNA complexes.

We have used molecular conjugates containing combinations of DNA, adenovirus, polylysine, and transferrin to transfect primary cells derived from canines with hemophilia B (factor IX deficiency), as well as a canine epithelial cell line. Transfection of canine hemophilia B fibroblasts with molecular conjugates resulted in efficient transfection and expression of luciferase DNA-adenovirus-polylysine (AdpL) conjugates or luciferase DNA-adenovirus-polylysine-transferrin (hTfpL/AdpL) conjugates. No expression in canine hemophilia B fibroblasts was evident after exposure to DNA alone, or DNA conjugated with polylysine and transferrin. Transfection efficiencies of 50% or more could be demonstrated in cells transfected with a beta-galactosidase reporter gene as part of an hTfpL/AdpL molecular conjugate. Transfection with canine factor IX AdpL conjugates or canine factor IX hTfpL/AdpL conjugates resulted in factor IX expression for more than 2 weeks in vitro in hemophilia B canine fibroblasts. Maximum levels of expression of over 700 ng of canine factor IX/10(6) cells/24 hr were demonstrated in fibroblasts after transfection with canine factor IX hTfpL/AdpL conjugates. Similar conjugates were used to transfect hemophilia B canine bone marrow stromal cells and Madin-Darby canine kidney cells that also expressed canine factor IX. The use of molecular conjugates to transfect primary cells may be feasible as a means of in vitro or in vivo gene therapy for hemophilia B, and can be tested in the canine hemophilia B model.

Adenoviridae↗
PubMed1998

Reduced invasive and metastatic potentials of KAI1-transfected melanoma cells.

KAI1 is a metastasis suppressor gene for human prostate cancer. To reveal the effect of KAI1 on the in vivo metastasis of tumors other than prostatic cancer, we transfected a human KAI1 cDNA into highly metastatic B16-BL6 murine melanoma cells and established stable transfectant clones with different expression levels of KAI1 message. The following results were obtained with the use of those transfectants. (1) Cell aggregation assay revealed a significantly enhanced Ca(2+)-independent aggregation of B16-BL6 cells by KAI1 cDNA transfection compared with mock transfectants (P < 0.01). (2) The in vivo phagokinetic activity and invasive ability of KAI1 transfectants were clearly decreased as compared with those of mock transfectants (P < 0.01). There was no significant effect of KAI1 expression on the in vitro or in vivo proliferation of B16-BL6 cells. (3) Lung colony formation of intravenously injected KAI1 transfectants in nude mice was significantly reduced as compared with mock transfectants or parental B16-BL6 cells (P < 0.01). These data suggest that KAI1 expression gives rise to the suppression of invasive and metastatic potentials of B16-BL6 cells.

Animals↗
PubMed1992

Replication of nodamura virus after transfection of viral RNA into mammalian cells in culture.

Nodamura virus (NOV) was purified from the hind limbs of infected suckling mice and used as a source of the two genomic RNAs of the virus, RNA 1 and RNA 2. Upon transfection of the viral RNAs into baby hamster kidney (BHK21) cells in culture, vigorous RNA replication ensued and single-stranded RNAs 1 and 2 accumulated to reach an abundance which approximated that of the cellular rRNAs. Transient synthesis of a small subgenomic RNA (RNA 3) was also observed, and double-stranded versions of RNAs 1, 2, and 3 were detected. Three major viral proteins were synthesized in transfected cells. Protein A (about 115 kDa) and protein B (about 15 kDa) were made transiently at early times after transfection, whereas a large amount of protein alpha (43 kDa), the precursor to the two viral coat proteins, was made continuously starting later in the infectious cycle. When very low concentrations of viral RNAs were used for transfection, preferential replication of RNA 1 occurred. This result was attributed to segregation of the transfected viral RNAs to separate cells in culture and the subsequent replication and amplification of RNA 1 in cells that had received no RNA 2. Accordingly, multiple passages of the viral RNAs by transfection at the limit dilution resulted in the purification of RNA 1 free of RNA 2 and demonstrated that RNA 1 was capable of prolonged autonomous replication which was also accompanied by the continuous synthesis of RNA 3. In cells transfected with RNA 1 alone, protein alpha was not synthesized and proteins A and B were made continuously. Electron microscopic analysis of BHK21 cells 24 h after transfection with NOV RNAs 1 and 2 showed that large numbers of virus particles accumulated in the cytoplasm and formed paracrystalline arrays in some regions. Whole NOV purified from transfected BHK21 cells was infectious for suckling mice and had an electrophoretic mobility that was similar but not identical to that of NOV purified from infected mouse muscle. The high yield of NOV, its simple genetic composition, and its unusual genome strategy make this virus an attractive system for the study of viral RNA replication in animal cells.

Animals↗
PubMed1985

Transfection by genomic DNA of cytochrome P1-450 enzymatic activity and inducibility.

An aryl hydrocarbon hydroxylase (AHH)-deficient gene A- mutant of the mouse line Hepa-1 was treated with calcium phosphate precipitates of DNA from Hepa-1, the rat line H4IIEC3, or an A- -human hybrid in which the A- mutation is complemented by the corresponding human gene. AHH+ transfectants were isolated by selection with benzo[ghi]perylene plus near UV. In addition, a gene A- mutant which also carries a mutation for hypoxanthine phosphoribosyltransferase deficiency was treated with the above genomic DNAs together with pSV2-gpt DNA, and cotransfectants were isolated after treatment with both benzo[ghi]pereylene and HAT. All transfectants and cotransfectants were inducible for AHH by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Both transfectants and cotransfectants were unstable during culture, rapidly losing AHH activity. Rat DNA-derived transfectants were probed in Southern blots with a cDNA probe to mouse cytochrome P1-450 that cross-hybridizes to the corresponding rat gene. All rat DNA-derived transfectants contained the rat P1-450 gene. In half of the transfectants, the rat gene was amplified four- to sevenfold. In one transfectant, the rat gene was truncated at the 3' end. The proportion of rat DNA in different transfectants, as determined by hybridization to a rat repetitive sequence, ranged from less than 1% to 5%. AHH activity and the rat P1-450 gene segregated together in subclones of one of the transfectants. These results demonstrate that the A gene is either the structural gene for cytochrome P1-450, or another very closely linked gene. Previous results (O. Hankinson et al., J. Biol. Chem. 260:1790-1795, 1985) favor the former alternative.

Animals↗
PubMed1997

Stable transfection of Acanthamoeba.

The promoter activity of an Acanthamoeba polyubiquitin gene was analyzed in its homologous system. A modified calcium phosphate transfection method using a neomycin marker vector was developed to achieve highly efficient transfection of the Acanthamoeba polyubiquitin gene into Acanthamoeba cells. In this transfection procedure, the calcium phosphate-DNA complex was formed gradually in the medium during incubation with cells and precipitated on the cells. The crucial factors for obtaining efficient transfection were the pH (6.95) of the transfection buffer used for the calcium phosphate precipitation and the amount (25 micrograms/96-well tissue culture plate) and form (circular) of transfecting DNA. Under these conditions, Acanthamoeba isolate 1B6 was transfected at an efficiency of about 40% with the constructed vector pOPSBU, a pOP13CAT-based polyubiquitin gene incorporated neomycin resistance vector. Acanthamoeba polyphaga was transfected at an efficiency of about 10% with this vector. Transfection of both Acanthamoeba strains appeared to result in low copy plasmid integration (about two copies per cell are suggested). The chloramphenicol acetyltransferase (CAT) assays showed that the promoter of the Acanthamoeba polyubiquitin gene in the constructed vector was especially strong in A. polyphaga, thus the pOPSBU-Acanthamoeba system may be useful for the construction of cDNA expression libraries, as well as for the expression of cloned genes.

Acanthamoeba↗
PubMedDate not supplied

Transient transfection of porcine granulosa cells after 3D culture in barium alginate capsules.

Three-dimensional culture systems in barium alginate capsules can be employed to maintain primary granulosa cells in an undifferentiated state for almost 6 days. This is due to a self-organization of cells in a pseudofollicular structure. The transfection of primary granulosa cells is a necessary condition when employing these culture systems for several purposes, for example as an in vitro toxicity test or the development of oocytes or zygotes. In this work, the feasibility of two transient transfection techniques (liposome-mediated and electroporation) was assessed in primary porcine granulosa cells after a 6-day culture in an artificial extracellular matrix (barium alginate membrane). Human recombinant green fluorescent protein was chosen as a molecular readout, and protein expression was assessed after 48 hours from transfection. Liposome-mediated transfection gave low transfection levels, with increasing yields from 2 to 12 microgDNA/ml of medium; the maximum percentage (85.7%) was reached at 12 microgDNA/ml of medium. Electroporation-mediated transfection yields were higher: the best results (81.7% of transfected cells) were achieved with two 50V pulses and 12 microg/ml DNA. The application of a single or double pulse (50V) at 4 mgDNA/ml gave negligible results. These results indicate that primary granulosa cell cultured in barium alginate capsules can be transfected by electroporation with high transfection yields.

Alginates↗
PubMed2004

Insulin receptor activation and down-regulation by cationic lipid transfection reagents.

BACKGROUND: Transfection agents comprised of cationic lipid preparations are widely used to transfect cell lines in culture with specific recombinant complementary DNA molecules. We have found that cells in culture are often resistant to stimulation with insulin subsequent to treatment with transfection agents such as LipofectAMINE 2000 and FuGENE-6. This is seen with a variety of different readouts, including insulin receptor signalling, glucose uptake into muscle cells, phosphorylation of protein kinase B and reporter gene activity in a variety of different cell types RESULTS: We now show that this is due in part to the fact that cationic lipid agents activate the insulin receptor fully during typical transfection experiments, which is then down-regulated. In attempts to circumvent this problem, we investigated the effects of increasing concentrations of LipofectAMINE 2000 on insulin receptor phosphorylation in Chinese hamster ovary cells expressing the human insulin receptor. In addition, the efficiency of transfection that is supported by the same concentrations of transfection reagent was studied by using a green fluorescent protein construct. Our data indicate that considerably lower concentrations of LipofectAMINE 2000 can be used than are recommended by the manufacturers. This is without sacrificing transfection efficiency markedly and avoids the problem of reducing insulin receptor expression in the cells. CONCLUSION: Widely-used cationic lipid transfection reagents cause a state of insulin unresponsiveness in cells in culture due to fully activating and subsequently reducing the expression of the receptor in cells. This phenomenon can be avoided by reducing the concentration of reagent used in the transfection process.

3T3-L1 Cells↗
PubMed2004

Anionic liposomal delivery system for DNA transfection.

The present study investigates the use of novel anionic lipoplexes composed of physiological components for plasmid DNA delivery into mammalian cells in vitro. Liposomes were prepared from mixtures of endogenously occurring anionic and zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), respectively, at a molar ratio of 17:83 (DOPG:DOPE). Anionic lipoplexes were formed by complexation between anionic liposomes and plasmid DNA molecules encoding green fluorescence protein (GFP) using Ca2+ ions. Transfection and toxicity were evaluated in CHO-K1 cells using flow cytometry and propidium iodide staining, respectively. Controls included Ca2+-DNA complexes (without lipids), anionic liposomes (no Ca2+), and a cationic liposomal formulation. Efficient delivery of plasmid DNA and subsequent GFP expression was achieved using anionic lipoplexes. Transfection efficiency increased with Ca2+ concentration up to 14 mM Ca2+, where transfection efficiency was 7-fold higher than in untreated cells, with minimum toxicity. Further increase in Ca2+ decreased transfection. Transfection efficiency of anionic lipoplexes was similar to that of cationic liposomes (lipofectAmine), whereas their toxicity was significantly lower. Ca2+-DNA complexes exhibited minimal and irregular transfection with relatively high cytotoxicity. A model was developed to explain the basis of anionic lipoplex uptake and transfection efficacy. Effective transfection is explained on the formation of nonbilayer hexagonal lipid phases. Efficient and relatively safe DNA transfection using anionic lipoplexes makes them an appealing alternative to be explored for gene delivery.

Animals↗
PubMed1983

Differences in the efficiency and stability of gene expression after transfection and nuclear injection: a study with a chick delta-crystallin gene.

The efficiency of an exogenous gene's expression was compared after its transfection and injection into various mouse cells to systematically evaluate these two gene transfer techniques. Special attention was paid to the period of transient expression. The gene used was a derivative of chicken delta-crystallin gene with the 5' end region replaced by a promoter base sequence of a retrovirus. Nuclear injection was more efficient than transfection in several respects: it was roughly one thousand times more efficient in producing gene-expressing cells than the transfection technique; it produced positive cells in every challenged cell line in contrast to the results of some unsuccessful trials found with transfection; and the maximum expression of the exogenous gene in a gene-transferred cell was much higher after injection than after transfection. With the transfection technique, use of a DNA-calcium phosphate coprecipitate was slightly more efficient than the use of DEAE-dextran. The stability of gene expression in transfected and nuclear-injected cells differed greatly: Expression of the exogenous gene in transfected cells was transmitted to 92% of the daughter cells per division, whereas its expression in injected cells was transmitted to only 32% of the daughter cells. This great difference in stability probably reflects different states of the major fraction of the exogenous gene: integration into chromosomes in transfected cells versus extrachromosomal localization in injected cells.

Animals↗
PubMed2006

Stearylated octaarginine and artificial virus-like particles for transfection of siRNA into primary rat neurons.

RNA interference (RNAi) provides a powerful experimental tool for sequence-specific gene silencing, allowing efficient analysis of gene function in a multitude of cell types. However, application of RNAi in primary mammalian neurons has been limited by low-transfection efficiency and considerable toxicity of conventional transfection methods. In this study, we evaluated a peptide-mediated and a polymer/lipid-based cellular delivery method for siRNA into rat primary neurons and compared the results with a commonly used liposomal transfection reagent. Stearylated octaarginine (Stearyl-R8) was used as polypeptide and artificial virus-like particles (AVPs) were used as a combined liposomal-polymeric vector, since both reagents have been previously shown to successfully transfect DNA into cell lines. Stearyl-R8 and AVPs both promoted siRNA transfection into primary hippocampal neurons via the endosomal pathway. SiRNA-mediated gene silencing could be effectively induced in primary neuron cultures. In comparison with the commonly used cationic liposome transfection agent, both novel reagents were less detrimental to cell metabolic activity. We conclude that these novel transfection methods yield performances comparable to cationic liposome-mediated transfection for siRNA, while being less cytotoxic in primary neurons. Stearyl-R8 and AVPs may therefore represent novel and more cost-efficient alternatives to conventional siRNA-transfection reagents.

Animals↗
PubMed1997

Optimization of transfection of human endothelial cells.

Recently developed transfection methods for mammalian cells provide a powerful means for the study of gene function. Unfortunately, human endothelial cells were relative refractory to the classic transfection techniques. In this study we compared the usability of calcium phosphate, DEAE-dextran transfection, transferrinfection, lipofection, and electroporation for the transfection of early passage HUVECs and for the human endothelial cell lines ECV 304 and EA.hy 926. The classic transfection methods resulted in no or only marginal expression of the reporter gene E. coli beta-galactosidase. For lipofection experiments we compared the commercially available liposome formulations DOTAP and Transfectam with liposomes prepared of dimethyldioctadecylammoniumbromide (DDAB) or 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE) as the cationic lipid compound and dioleylphosphatidylethanolamine (DOPE) or Azolectin (a crude fraction of soybean lipids, commercially available as phosphatidylcholine II) as neutral co-lipid. Because the protocol for the chemical synthesis of DMRIE has not been published yet, we developed a protocol for the chemical synthesis of this cationic lipid. With transfection protocols optimized for each cell line, we could achieve transfection efficiencies up to 2%. Compared to the other methods used, the lipofection proved to be a reliable technique for the efficient transfection of the human endothelial cell lines ECV 304 and EA.hy 926. Although we achieved a maximum transfection efficiency of 0.45% for the lipofection of HUVEC, the electroporation seemed to be the better choice for these cells.

Cell Line↗
PubMed2000

Induction of antitumor immunity by vaccination of dendritic cells transfected with MUC1 RNA.

Dendritic cells (DC) are potent APCs. In this study, murine bone marrow-derived DC were transfected with RNA encoding the MUC1 Ag that is aberrantly overexpressed in human breast and other carcinomas. The MUC1 RNA-transfected DC exhibited cell surface expression of MUC1 and costimulatory molecules. After injection at the base of the tail, the transfected DC were detectable in inguinal lymph nodes by dual immunochemical staining. Vaccination of wild-type mice with MUC1 RNA-transfected DC induced anti-MUC1 immune responses against MUC1-positive MC38/MUC1, but not MUC1-negative, tumor cells. Mice immunized with the transfected DC were protected against challenge with MC38/MUC1 tumor cells. Furthermore, mice with established MC38/MUC1 tumors were eliminated after receiving the vaccination. CTLs isolated from mice immunized with the transfected DC exhibited specific cytolytic activity against MC38/MUC1 tumor cells. In contrast to these findings, there was little if any anti-MUC1 immunity induced with the transfected DC in MUC1 transgenic (MUC1.Tg) mice. However, coadministration of the transfected DC and IL-12 reversed the unresponsiveness to MUC1 Ag in MUC1.Tg mice and induced MUC1-specific immune responses. These findings demonstrate that vaccination of DC transfected with MUC1 RNA and IL-12 reverses tolerance to MUC1 and induces immunity against MUC1-positive tumors.

Animals↗
PubMed2000

lacZ-neoR transfected glioma cells in syngeneic rats: growth pattern and characterization of the host immune response against cells transplanted inside and outside the CNS.

The rat glioma cell lines BT4C and BT4Cn were stably transfected with the bacterial lacZ-neomycin resistance (neoR) gene construct. Both transfected (BT4ClacZ and BT4CnlacZ) and untransfected cell lines were injected intracerebrally and subcutaneously into rats. Survival time, morphology, growth rate and immunological properties of the tumors were studied. Survival time was significantly prolonged after intracerebral implantation of the transfected cell lines. No similar response was found in nude rats, indicating an immunological response towards the lacZ-neoR-transfected cells in immunocompetent animals. Morphological observations showed that the lacZ-neoR-transfected gliomas were smaller and had a distinct boundary with the normal brain tissue, whereas the parental cell lines revealed a more diffuse growth pattern. Immunostaining showed a higher proportion of immunocompetent cells infiltrating the lacZ-neoR-transfected tumors. After s.c. injection, the lacZ-neoR-transfected BT4C cell line had a prolonged lag phase before assuming a growth rate similar to that of the parental cells. The BT4CnvlacZ tumors initially grew fastest, but then disappeared within 3 weeks. A similar response was observed with mock-transfected tumor cells. A (3)HTdR-incorporation assay on spleen cells from rats transplanted s.c. with BT4CnvlacZ cells showed a 10-fold increase in cell activation as compared with rats with BT4Cn tumors. A humoral response towards the transfected cells was verified by Western-blot analyses.

Animals↗
PubMed2000

[The influence of hTGFbeta(1) gene transfection on the proliferation of NIH 3T3 fibroblast].

OBJECTIVE: To investigate the influence of hTGFbeta(1) ene transfection on the proliferation of in vitro cultured NIH 3T3 fibroblast. METHODS: Lipofictin mediated co-transfected method was employed to transfect hTGFbeta(1) plasmid DNA into NIH 3T3 fibroblasts of the rats. Cell growth counting method, MTT, flow cytometry (FCM) and soft agar clone forming method were adopted to examine the biology of the transfected cells. RESULTS: (1) Fibroblast growth slowed down after hTGFbeta(1) transfection, especially on 4th to 6th days after the transfection, which was accompanied by decreased synthesis of DNA as indicated by the increase of G(1) phase percentage (from 39.9% to 66.2% P < 0.05) and the decrease of S phase percentage (from 40.2% to 26.8%, P < 0.05); (2) There was no evident change of all the phases in a cell cycle in the blank load transfection group and in control group. (3) There was a good correlation between MTT and cell counting method (co-efficient 0.992) (4) The formation rate of the soft agar clone of fibroblast decreased from 1.18% to 0.55% (P < 0.05) after hTGFbeta(1) transfection. CONCLUSION: hTGFbeta(1) gene transfection could inhibit fibroblast proliferation, which might be related to the interference of TGFbeta(1) with the DNA synthesis of fibroblasts.

3T3 Cells↗
PubMed2002

[Functional effects of dendritic cells transfected with Epstein-Barr virus latent membrane protein 2A recombinant adenovirus].

BACKGROUND: To study the effects of dendritic cells (DC) transfected with recombinant adenovirus encoding Epstein-Barr virus(EBV)latent membrane protein 2A(LMP2A)gene, and to provide evidence for further investigation on the therapeutic vaccine against EBV-associated malignancies. METHODS: DCs were transfected with EBV-LMP2A recombinant adenovirus (Ad5-LMP2A), which was generated by homologous recombination. The expression of LMP2A protein on mature DC transfected with Ad5-LMP2A at different multiplicity of infection(MOI)was analyzed by fluorescence activated cell sorting(FACS), and the dead cells were counted by trypan blue staining. The alteration of surface markers on mature DC including CD1a, CD83, CD40, CD80, HLA?DR was detected by means of FACS before and after transfection. Meanwhile, the functions including stimulating allogenetic T cells reaction and expressing IL12 P40 mRNA on transfected DC were measured by methods of 3H-thymidine uptake and fluorescent semi?quantitative polymerase chain reaction (PCR), respectively. RESULTS: About 80% mature DC expressed LMP2A protein and >92% cells were viable after transfection at the MOI of 200 No. significant changes in the surface markers and the cytomorphology of mature DCs were detected during the transfection. Transfected DC still have strong potential to stimulate the proliferation of allogenetic T cells and to express IL-12 P40 mRNA. CONCLUSION: EBV-LMP2A gene,which was carried by adenovirus vectors, could be transferred into DC with high efficiency. The function of mature DC was not affected significantly by the transfection of Ad5-LMP2A.

Adenoviridae↗
PubMed2002

Gene transfection and expression of transforming growth factor-beta1 in nonobese diabetic mouse islets protects beta-cells in syngeneic islet grafts from autoimmune destruction.

Nonobese diabetic (NOD) mice develop diabetes and destroy syngeneic islet grafts through an autoimmune response. Because transforming growth factor (TGF)-beta1 downregulates immune responses, we tested whether overexpression of TGF-beta1 by gene transfection of NOD mouse islets could protect beta-cells in islet grafts from autoimmune destruction. NOD mouse islet cells were transfected with an adenoviral DNA expression vector encoding porcine latent TGF-beta1 (Ad TGF-beta1) or the adenoviral vector alone (control Ad vector). The frequency of total islet cells expressing TGF-beta1 protein was increased from 12 +/- 1% in control Ad vector-transfected cells to 89 +/- 4% in Ad TGF-beta1-transfected islet cells, and the frequency of beta-cells that expressed TGF-beta1 was increased from 12 +/- 1% to 60 +/- 7%. Also, secretion of TGF-beta1 was significantly increased in islets that overexpressed TGF-beta1. Ad TGF-beta1-transfected NOD mouse islets that overexpressed TGF-beta1 prevented diabetes recurrence after transplantation into diabetic NOD mice for a median of 22 days compared with only 7 days for control Ad vector-transfected islets (p = 0.001). Immunohistochemical examination of the islet grafts revealed significantly more TGF-beta1+ cells and insulin+ cells and significantly fewer CD45+ leukocytes in Ad TGF-beta1-transfected islet grafts. Also, islet beta-cell apoptosis was significantly decreased whereas apoptosis of graft-infiltrating leukocytes was significantly increased in Ad TGF-beta1-transfected islet grafts. These observations demonstrate that overexpression of TGF-beta1, by gene transfection of NOD mouse islets, protects islet beta-cells from apoptosis and autoimmune destruction and delays diabetes recurrence after islet transplantation.

Animals↗