RESUMO
Human factor IX is synthesized in the liver and secreted in the blood, where it participates in a group of reactions involving coagulation factors and proteins that permit sanguinary coagulation. In this work two lines of transgenic mice were developed to express the FIX gene in the mammalian glands under control of milk beta-casein promoter. The founding females secreted the FIX in their milk (3% total soluble protein). The stable integration of transgene was confirmed by southern blot analysis. The presence of the FIX recombinant protein in the milk of transgenic females was confirmed by western blot and the clotting activity was revealed in blood-clotting assays. The coagulation activity in human blood treated with recombinant FIX increased while the time of coagulation decreased. Our results confirm the production of a large amount of recombinant biologically active FIX in the mammary gland of transgenic mice.
Assuntos
Fator IX/biossíntese , Glândulas Mamárias Animais/metabolismo , Proteínas do Leite/biossíntese , Animais , Southern Blotting , Western Blotting , Fator IX/metabolismo , Fator IX/fisiologia , Feminino , Lactação , Masculino , Camundongos , Camundongos Transgênicos , Proteínas do Leite/genética , Proteínas do Leite/metabolismo , Tempo de Tromboplastina Parcial , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismoRESUMO
Generation of transgenic animals is invaluable for both basic and applied research, as it enables the production of biologically active proteins and immunologically compatible organs for xenotransplantation, improvement of livestock production traits, and establishment of animal models of human disease. However, transgene expression is commonly highly variable, even among cell lines independently transformed with the same construct. Consequently, a great number of transfections and screening is needed to achieve transgenic cell lines showing expected phenotype. In this study, we sequenced transgene-host DNA junctions of transgene integration loci in 26 independently transformed Madin Darby bovine kidney (MDBK) cell lines produced by direct liposome transfection. For 15 rescued clones, sequences were of sufficient length and quality to determine unambiguously the position of integration in the bovine genome. Results revealed that transgenes were integrated in 12 different chromosomes, suggesting that there was no chromosomal preference for insertion of exogenous DNA. Most integration events occurred into transcriptionally active regions. No correlation was found between integration into transcribed sequences and the expression level of the beta-gal transgene.
Assuntos
Engenharia Genética/métodos , Técnicas Genéticas , Rim/citologia , Transgenes , Animais , Sequência de Bases , Bovinos , Técnicas de Cultura de Células/métodos , DNA/metabolismo , Expressão Gênica , Dados de Sequência Molecular , Transcrição Gênica , Transfecção , beta-Galactosidase/metabolismoRESUMO
An association of two techniques, nuclear transfer (NT), and transfection of somatic animal cells, has numerous potential applications and considerable impact, mainly in agriculture, medicine, pharmacy, and fundamental biology. In addition, somatic cell nuclear transfer is the most efficient alternative to produce large transgenic animals. We compared in vitro and in vivo developmental capacities of NT using fibroblast cells isolated from a 14-month-old cloned Simmental heifer (FCE) vs the same line transfected with a plasmid containing neomycin-resistant genes (TFCE). There were no significant differences (P > 0.5) in either fusion (116/149 = 78% vs 216/301 = 72%), cleavage (78/116 = 67% vs 141/216 = 65%) and blastocyst (35/116 = 30% vs 52/216 = 24%) rates or in pregnancy rate at 30 to 35 days after embryo transfer (2/17 vs 3/17) between NT using FCE and TFCE, respectively. Transfection and long-term in vitro culture of transfected cells did not affect developmental capacity of NT embryos up to 40 days of gestation.
