RESUMO
Testis-mediated gene transfer (TMGT) has been used as in vivo gene transfer technology to introduce foreign DNA directly into testes, allowing mass gene transfer to offspring via mating. In this study, we used plasmid DNA (pEGFP-N1) mixed with dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMA) or liposome (Lipofectin) in an attempt to improve TMGT. Males receiving consecutive DNA complex injections were mated to normal females to obtain F0 progeny. In vivo evaluation of EGFP expression, RT-PCR and PCR were used to detect the expression and the presence of exogenous DNA in the progeny. We also evaluated possible testicular damage by histological procedures. PC R and RT-PCR analyses revealed that liposome and DMSO increased the rate of TMGT. Histological analyses demonstrated that repeated (4 times) injections of DNA complexes can affect spermatogenesis. DMSO was the most deleterious among the reagents tested. In this study, we detected the presence of transgene in the progeny, and its expression in blood cells. Consecutive injections of DNA complexes were associated with impaired spermatogenesis, suggesting requirement of optimal conditions for DNA delivery through TMGT.
Assuntos
Animais , Feminino , Camundongos , Dimetil Sulfóxido/farmacologia , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/administração & dosagem , Camundongos Transgênicos/genética , Testículo , Transgenes , Animais Geneticamente Modificados , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Lipossomos/farmacologia , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Testículo/efeitos dos fármacos , Testículo/patologia , Transfecção/métodosAssuntos
Humanos , Animais , Expressão Gênica/genética , Fatores de Transcrição/genética , Biologia Molecular/tendências , Transcrição Gênica/genética , Transferrina/genética , Adenovírus Humanos/genética , Carcinoma Hepatocelular/genética , Expressão Gênica/fisiologia , Fatores de Transcrição/fisiologia , Camundongos Transgênicos/genética , Células de Sertoli , Transcrição Gênica/fisiologiaRESUMO
Transgenic mice and rabbits were generated using a chimeric gene comprising the human erythropoietin (hEPO) cDNA under the 5' and 3' regulatory sequences of the rabbit whey acidic protein gene. Transgenic mice expressed hEPO at levels of 0.01 mg/l in the milk of lactating females showing that the genetic construct was functional. Reverse transcriptase polymerase chain reaction with RNA from various tissues showed that this transgene was expressed mainly in the ovary and mammary gland. In rabbits, we demonstrated the germ line transmission of the transgene. The hEPO was obtained in the milk of lactating females at levels of up to 0.0003 mg/l. Although the expression levels were low, biologically active hEPO was obtained in the milk of transgenic rabbits without any apparent detrimental effect for the animals. In vitro, the specific activity of the rabbit-derived hEPO was higher than that reported for the natural hEPO, thus suggesting differences in the glycosylation pattern in at least part of the molecules secreted by the mammary gland of transgenic rabbits