RESUMO
We report here a very efficient method for the in vivo transfer of therapeutic plasmid DNA into porcine muscle fibers by using electric pulses of low field intensity. We evaluated delivery of 0.1-3 mg of plasmid vectors that encode reporter secreted-embryonic alkaline phosphatase (SEAP) or therapeutic growth hormone releasing hormone (GHRH). Reporter gene studies showed that internal needle electrodes give a 25-fold increase in expression levels compared with caliper electrodes in skeletal muscle in swine. Dose and time courses were performed. Pigs injected with 0.1 mg plasmid had significantly greater weight gain than controls over 53 days (22.4 +/- 0.8 kg vs. 19.7 +/- 0.03 kg, respectively; P<0.01). The group treated with GHRH-expressing plasmid at 14 days of age demonstrated greater weight gain than controls at every time point (25.8 +/- 1.5 kg vs. 19.7 +/- 0.03 kg; P<0.01). Body composition studies by dual X-ray absorbitometry showed a 22% decrease in fat deposition (P<0.05) and a 10% increase in bone mineral density (P<0.004). Our studies demonstrate that by optimizing the electroporation method, favorable physiological changes, such as enhanced weight gain and improved body composition, can be obtained at extremely low plasmid doses in a large mammal.
Assuntos
Eletroporação/métodos , Vetores Genéticos , Hormônio Liberador de Hormônio do Crescimento/genética , Músculo Esquelético , Plasmídeos , Animais , Composição Corporal , Técnicas de Transferência de Genes , Cinética , Proteínas/genética , Suínos , Aumento de PesoRESUMO
Regulated animal growth occurred following a single electroporated injection of a mixture of two plasmids (10 microg of DNA), one expressing the GeneSwitch regulator protein, the other an inducible growth hormone releasing hormone (GHRH) gene, into the tibialis anterior muscles of adult SCID mice. Administration of the ligand mifepristone (MFP) up-regulated GHRH expression, as shown by elevations of IGF-I levels, and when MFP dosing was withdrawn, IGF-I returned to baseline levels. Five cycles of IGF-I induction were observed during a five-month period. Chronic MFP dosing for 25 days increased lean body mass, weight gain, and bone mineral density significantly compared with non-MFP treated controls. In summary, long-term drug-regulated GHRH expression was achieved following plasmid-based gene therapy, and chronic induction of GHRH expression in adult animals led to improvements in weight gain and body composition.
Assuntos
Vetores Genéticos , Hormônio Liberador de Hormônio do Crescimento/genética , Mifepristona/farmacologia , Músculo Esquelético , Aumento de Peso , Animais , Composição Corporal , Densidade Óssea , Células Cultivadas , Galinhas , Terapia Genética , Vetores Genéticos/administração & dosagem , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Injeções , Fator de Crescimento Insulin-Like I/biossíntese , Cinética , Ligantes , Camundongos , Camundongos SCID , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Plasmídeos , Engenharia de Proteínas/métodos , Ativação TranscricionalRESUMO
The im electroporated injection of a protease-resistant GH-releasing hormone cDNA into rat dams at 16 d gestation resulted in enhanced long-term growth of the F(1) offspring. The offspring were significantly heavier by 2 wk of age, and the difference was sustained to 10 wk of age. Consistent with their augmented growth, the plasma IGF-I concentration of the F(1) progeny was increased significantly. The pituitary gland of the offspring was significantly heavier and contained an increased number of somatotrophs and PRL-secreting cells, which is indicative of modification of cell lineage differentiation. These unique findings demonstrate that enhanced GH-releasing hormone expression in pregnant dams can result in intergenerational growth promotion by altering development of the pituitary gland in the offspring.