RESUMO
The mammary gland serves as a valuable bioreactor system for the production of recombinant proteins in lactating animals. Pharmaceutical-grade recombinant protein can be harvested from the milk of transgenic animals that carry a protein of interest under the control of promoter regions genes encoding milk proteins. Whey acidic protein (WAP), for example, is predominantly expressed in the mammary gland and is regulated by lactating hormones during pregnancy. We cloned the 5'-flanking region of the porcine WAP gene (pWAP) to confirm the sequence elements in its promoter that are required for gene-expression activity. In the present study, we investigated how lactogenic hormones--including prolactin, hydrocortisone, and insulin--contribute to the transcriptional activation of the pWAP promoter region in mammalian cells, finding that these hormones activate STAT5 signaling, which in turn induce gene expression via STAT5 binding sites in its 5'-flanking region. To confirm the expression and hormonal regulation of the 5'-flanking region of pWAP in vivo, we generated transgenic mice expressing human recombinant granulocyte colony stimulating factor (hCSF2) in the mammary gland under the control of the pWAP promoter. These mice secreted hCSF2 protein in their milk at levels ranging from 242 to 1,274.8 ng/ml. Collectively, our findings show that the pWAP promoter may be useful for confining the expression of foreign proteins to the mammary gland, where they can be secreted along with milk.
Assuntos
Glândulas Mamárias Animais/metabolismo , Proteínas do Leite/biossíntese , Leite/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Animais , Feminino , Humanos , Lactação , Camundongos , Proteínas do Leite/genética , Gravidez , Fator de Transcrição STAT5/genética , SuínosRESUMO
The laying hen is the best model for oviduct growth and development. The chicken oviduct produces the egg components, including the egg white and eggshell. However, the mechanism of egg component production during oviduct development requires further investigation. Vitelline membrane outer layer protein 1 (VMO-1) is found in the outer layer of the vitelline membrane of avian eggs. Comparison of the chicken VMO-1 protein-coding sequence and the human, mouse, rat, and bovine VMO-1 proteins via multiple sequence alignment analysis revealed high degrees of homology of 55%, 53%, 48%, and 54%, respectively. Although the avian homologue of VMO-1 is highly expressed in the magnum of the oviduct, little is known about the transcriptional and posttranscriptional regulation of VMO-1 during oviduct development. The results of this study revealed that estrogen induces VMO-1 messenger RNA (mRNA) expression in oviduct cells in vitro. The expression of genes interacting with VMO-1 by RNA interference (RNAi) functional analysis revealed that ovomucin expression was decreased by VMO-1 silencing. In addition, gga-miR-1623, 1552-3p, and 1651-3p influenced VMO-1 expression via its 3'-UTR, suggesting the posttranscriptional regulation of VMO-1 expression in chickens. Collectively, these results suggest that VMO-1 is an estrogen-induced gene that is posttranscriptionally regulated by microRNAs (miRNAs). The present study may contribute to an understanding of egg component production during chicken oviduct development.
Assuntos
Proteínas Aviárias/genética , Galinhas/crescimento & desenvolvimento , Galinhas/genética , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Oviductos/metabolismo , Membrana Vitelina/metabolismo , Envelhecimento/genética , Sequência de Aminoácidos , Animais , Proteínas Aviárias/metabolismo , Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Estradiol/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Genoma , MicroRNAs/genética , Dados de Sequência Molecular , Oviductos/efeitos dos fármacos , Oviductos/crescimento & desenvolvimento , Filogenia , Reprodutibilidade dos Testes , Alinhamento de Sequência , Análise de Sequência de Proteína , Transcrição Gênica/efeitos dos fármacos , Membrana Vitelina/efeitos dos fármacosRESUMO
Extracellular superoxide dismutase (EC-SOD) is a metalloprotein and functions as an antioxidant enzyme. In this study, we used lentiviral vectors to generate transgenic chickens that express the human EC-SOD gene. The recombinant lentiviruses were injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Of 158 injected embryos, 16 chicks (G0) hatched and were screened for the hEC-SOD by PCR. Only 1 chick was identified as a transgenic bird containing the transgene in its germline. This founder (G0) bird was mated with wild-type hens to produce transgenic progeny, and 2 transgenic chicks (G1) were produced. In the generated transgenic hens (G2), the hEC-SOD protein was expressed in the egg white and showed antioxidant activity. These results highlight the potential of the chicken for production of biologically active proteins in egg white.
