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1.
Xenotransplantation ; 16(6): 477-85, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20042047

RESUMO

BACKGROUND: Inhibition of the T-cell-mediated immune response is a necessary component of preventing rejection following xenotransplantation with pig alpha1,3-galactosyltransferase gene-knockout (GTKO) organs. Cytotoxic T lymphocyte-associated antigen (CTLA4) is a co-stimulatory molecule that inhibits T-cell activity and may be useful in prolonging graft rejection. METHODS: An expression vector was built containing the extracellular coding region of porcine (p) CTLA4 fused to the hinge and CH2/CH3 regions of human IgG1 (pCTLA4-Ig). Pigs transgenic for pCTLA4-Ig, on either a GTKO or wild-type (WT) genetic background, were produced by nuclear transfer and characterized using Western blot analysis, immunofluorescence, ELISA, and necropsy. RESULTS: Fifteen pCTLA4-Ig-transgenic piglets resulted from five pregnancies produced by nuclear transfer. All transgenic pigs exhibited robust expression of the pCTLA4-Ig protein and most expressed the transgene in all organs analyzed, with significant levels in the blood as well. Despite initial good health, these pigs exhibited diminished humoral immunity, and were susceptible to infection, which could be managed for a limited time with antibiotics. CONCLUSIONS: Viable pigs exhibiting robust and ubiquitous expression of pCTLA4-Ig were produced on both a WT and GTKO background. Expression of pCTLA4-Ig resulted in acute susceptibility to opportunistic pathogens due at least in part to a significantly compromised humoral immune status. As this molecule is known to have immunosuppressive activity, high levels of pCTLA4-Ig expression in the blood, as well as defective development related to exposure to pCTLA4-Ig in utero, may contribute to this reduced immune status. Prophylactic treatment with antibiotics may promote survival of disease-free transgenic pigs to a size optimal for organ procurement for transplantation. Additional genetic modifications and/or tightly regulated expression of pCTLA4Ig may reduce the impact of this transgene on the humoral immune system.


Assuntos
Animais Geneticamente Modificados , Imunoconjugados/genética , Imunossupressores/imunologia , Suínos/genética , Abatacepte , Animais , Feminino , Humanos , Imunidade Humoral/imunologia , Imunoconjugados/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Técnicas de Transferência Nuclear , Gravidez , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ovinos , Distribuição Tecidual , Transgenes , Transplante Heterólogo/imunologia
2.
Gene Expr ; 14(5): 251-63, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19630269

RESUMO

DNA methylation plays a significant role in the expression of the genetic code and affects early growth and development through their influence on gene expression. Manipulation of the DNA methylation marks of differentiated cells will allow a better understanding of the different molecular processes associated with chromatin structure and gene expression. The objective of this study was to identify small interfering RNAs (siRNAs) with the ability to reduce DNA methyltransferase 1 (Dnmt1) mRNA and consequently decrease Dnmt1 protein as well as DNA methylation in porcine cells. Fibroblasts from four porcine fetuses were established and cultured in 5% CO2 in air at 38 degrees C. Optimal transfection conditions were evaluated using a FITC-labeled control siRNA. Four Dnmt1-specific siRNAs were evaluated upon transfection of each cell line. A nonsilencing siRNA was used as a negative control. The expression patterns of Dnmt1 were analyzed by Q-PCR. The combination of 1 microg of siRNA and a 1:6 siRNA to transfection reagent ratio produced the highest transient transfection rates without affecting cell viability. Downregulation of Dnmt1 varied between siRNAs. Transfection of porcine cells with highly effective siRNAs resulted in a drastic reduction of Dnmt1 mRNA and a slight decrease in protein production. However, this small reduction in the protein concentration induced significant genomic hypomethylation. These data suggest that although Dnmt1 mRNA abundance plays an important role during protein regulation, Dnmt1 enzyme is mainly posttranscriptionally regulated. Subsequent use of these cells for cloning, differentiation, and cancer studies will provide insight as to how methylation of the DNA affects genomic reprogramming.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Fibroblastos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , DNA Metiltransferase 3A , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , RNA Mensageiro/metabolismo , Sus scrofa , Transfecção , DNA Metiltransferase 3B
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