RESUMO
Keratinocyte growth factor 1 (KGF1) is a growth factor that promotes epidermal cell proliferation, migration, differentiation, and wound repair. It is expressed at low levels in a form of inclusion body in E. coli. In order to increase its expression and activity, we produced tobacco plants expressing KGF1 via Agrobacterium-mediated transformation using a potato virus X (PVX)-based vector (pgR107). The vector contained the sequence encoding the KGF1 gene fused with a green florescence protein. The recombinant plasmid was introduced into leaf cells of Nicotiana benthamiana (a wild Australian tobacco) via Agrobacterium-mediated agroinfiltration. As determined by fluorescence and Western blot of leaf extracts, the KGF1 gene was correctly translated into the tobacco plants. The recombinant KGF1 was purified from plant tissues by heparin affinity chromatography, and cell proliferation in NIH/3T3 cells was stimulated by the purified KGF1. The purified KGF1 was also applied to the wounds of type-II diabetic rats. KGF1 had accumulated to levels as high as 530 µ g/g fresh weight in the leaves of agroinfected plants. We show that plant-derived KGF1 can promote the proliferation of NIH/3T3 cells and have significant effects on the type-II diabetic rat. The present findings indicated that KGF1 from tobacco maintains its biological activity, implying prospective industrial production in a plant bioreactor.
Assuntos
Proliferação de Células/efeitos dos fármacos , Fator 7 de Crescimento de Fibroblastos , Nicotiana , Plantas Geneticamente Modificadas , Cicatrização/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental , Feminino , Fator 7 de Crescimento de Fibroblastos/biossíntese , Fator 7 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/isolamento & purificação , Fator 7 de Crescimento de Fibroblastos/farmacologia , Humanos , Camundongos , Células NIH 3T3 , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Nicotiana/química , Nicotiana/genética , Nicotiana/metabolismoRESUMO
AIM: To construct a lentiviral vector carrying human nuclear distribution protein C (hNUDC)-specific shRNA (sh-NUDC-F) and knock down hNUDC expression in Dami cells by infection of the lentivirus. METHODS: After labeling of green fluorescent protein (GFP), sh-NUDC-F was cloned into lentiviral vector pRRL-cPPT-CMV-X-PRE-SIN, and the high-quality plasmid was transfected into 293T cells to produce lentiviral particles by the calcium phosphate method. After high-speed centrifugation, lentiviral particles were collected and determined for its titer. The high-titer lentiviral particles were then transduced into Dami cells. By detecting the expression of GFP in lentiviral vector using a microscope, the transduction efficiency was readily figured out. And hNUDC protein level was detected by Western blotting. RESULTS: hNUDC was totally knocked down by the efficient transduction of Dami cells with the lentivirus carrying sh-NUDC-F. CONCLUSION: Lentiviral vector containing sh-NUDC-F can be successfully packaged in 293T cells and then efficiently transduced into Dami cells to silence hNUDC gene.
Assuntos
Proteínas de Ciclo Celular/genética , Vetores Genéticos , Lentivirus/genética , Proteínas Nucleares/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ciclo Celular/metabolismo , Expressão Gênica , Inativação Gênica , Células HEK293 , Humanos , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução GenéticaRESUMO
AIM: Used RFP gene to construct a RNA interference vector for convenience to obtain the good effective hairpins sequence. METHODS: NUDC and RFP genes were cloned into pDs vector separately, resulting in pDs-NUDC- RFP. as above, human U6 promoter and 9 hairpins sequence of NUDC were cloned into the pDs- NUDC-RFP vector separately.The RNA interfererence vectors target to 9 points of NUDC were constructed. Construct- ed recombinant vectors and then were identified by restrictive digestion and DNA sequencing.293T cells were transfected with the recombinant DNA samples by the liposome complex method, and then fluorescence photographs were taken. RESULTS: Enzyme digestion and DNA sequencing showed that the target gene and shRNA fragments were correctly inserted in pDs vector. fluorescence photographs showed that shNUDC-A is the best effective fragment. CONCLUSION: The NUDC gene targeted shRNA and its vector are successfully constructed.
Assuntos
Fusão Gênica Artificial/métodos , Proteínas de Ciclo Celular/genética , Vetores Genéticos/genética , Proteínas Luminescentes/genética , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Células HEK293 , Humanos , Plasmídeos/genética , Plasmídeos/metabolismo , Mapeamento por Restrição , Proteína Vermelha FluorescenteRESUMO
Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.
