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1.
FASEB J ; 29(6): 2386-96, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25690652

RESUMO

Currently, transgenic animals have found a wide range of industrial applications and are invaluable in various fields of basic research. Notably, deposition of transgene-encoded proteins in the egg white (EW) of hens affords optimal production of genetically engineered biomaterials. In the present study, we developed a minisynthetic promoter modulating transgene transcription specifically in the hen's oviduct, and assayed the bioactivity of human epidermal growth factor (hEGF) driven by that promoter, after partial purification of epidermal growth factor (EGF) from transgenic hen eggs. Our minisynthetic promoter driving expression of chicken codon-optimized human epidermal growth factor (cEGF) features 2 consecutive estrogen response elements of the ovalbumin (OV) promoter, ligated with a 3.0 kb OV promoter region carrying OV regulatory elements, and a 5'-UTR. Subsequently, a 3'-UTR carrying the poly-A tail sequence of the OV gene was added after incorporation of the cEGF transgene. Finally, we partially purified cEGF from transgenic hen eggs and evaluated the biofunctional activities thereof in vitro and in vivo. In the in vitro assay, EW-derived hEGF exhibited a proliferative effect on HeLa cells similar to that of commercial hEGF. In the in vivo assay, compared to the nontreated control, transgenic hen egg-derived EGF afforded slightly higher levels of re-epithelialization (via fibroplasia) and neovascularization of wounded skin of miniature pigs than did the commercial material. In conclusion, transgenic hens may be used to produce genetically engineered bioactive biomaterials driven by an oviduct-specific minisynthetic promoter.


Assuntos
Galinhas/metabolismo , Clara de Ovo/química , Fator de Crescimento Epidérmico/metabolismo , Oviductos/metabolismo , Regiões Promotoras Genéticas/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Galinhas/genética , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/farmacologia , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Ovalbumina/genética , Pele/irrigação sanguínea , Pele/efeitos dos fármacos , Pele/lesões , Suínos , Porco Miniatura , Cicatrização/efeitos dos fármacos
2.
BMB Rep ; 43(11): 726-31, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21110915

RESUMO

We report the tissue-specific distribution of chitinolytic activity in Korean ginseng root and characterize two 31-kDa chitinolytic enzymes. These two enzymes (SBF1 and SBF2) were purified 70- and 81-fold with yields of 0.75 and 1.25%, respectively, and exhibited optimal pH and temperature ranges of 5.0-5.5 and 40-50(o)C. With [(3)H]-chitin as a substrate, K(m) and V(max) values of SBF1 were 4.6 mM and 220 mmol/mg-protein/h, respectively, while those of SBF2 were 7.14 mM and 287 mmol/mg-protein/h. The purified enzymes showed markedly less activity with p-nitrophenyl-N-acetylglucosaminide and fluorescent 4-methylumbelliferyl glycosides of D-N-acetylglucosamine oligomers than with [(3)H]-chitin. End-product inhibition of both enzymes demonstrated that both are endochitinases with different N-acetylglucosaminidase activity. Furthermore, the NH(2)-terminal sequence of SBF1 showed a high degree of homology with other plant chitinases whereas the NH(2)-terminal amino acid of SBF2 was blocked. [BMB reports 2010; 43(11):726-731].


Assuntos
Quitinases/química , Panax/enzimologia , Acetilglucosaminidase/metabolismo , Quitinases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Raízes de Plantas/enzimologia , República da Coreia , Especificidade por Substrato , Temperatura , Fatores de Tempo
3.
J Androl ; 30(6): 690-702, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19478331

RESUMO

Although chicken spermatogonial stem cells (SCs) are important in spermatogenesis and transgenic research, little is known about these cells. Recently, our group constructed an in vitro culture system to establish germline stem cells (GSCs). To examine the mechanism of chicken spermatogonial SC development, we constructed a proteome map of GSCs from 4-week-old chicken testes. Soluble extracts of the GSCs were fractionated by 2-dimensional gel electrophoresis (pH 4-7). Several protein spots, including those that displayed significantly high levels, were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Of the 82/250 GSC spots examined, 56 yielded mass spectra that matched avian proteins found in the on-line databases. All of the identified proteins were classified into functional groups. This type of proteome map is an important resource for research on spermatogenesis and transgenesis.


Assuntos
Proteoma , Espermatogônias/metabolismo , Animais , Células Cultivadas , Galinhas , Perfilação da Expressão Gênica , Masculino
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