Stable expression of human interlukin┐2gene in transfected keratinocyt
Stableexpressionofhumaninterlukin┐2geneintransfectedkeratinocytes,INTRODUCTION,MATERIALSANDMETHODS,Cellcultureandtransfection,RESULTS,DISCUSSION,REFER
Keywords:keratinocytes;gene therapy;human interlukin-2CLCnumber:R394.3
Abstract:AIM To investigate the stable expression and se-cretion of human interlukin-2(IL-2)gene in transfected ker-atinocytes and assess the possibility of keratinocytes as a tar-get cell for gene therapy.METHODS Primary epidermal keratinocytes were isolated,cultured in serum-free medium,transfected by Lipofectamine and selected with antibiotic G418.The expression and activity of IL-2were assessed with techniques of DNA dot blot,RNA dot blot,hybridization in situ,immunohistochemistry,Western blot and MTT colori-metric assay.RESULTS Positive signal was found in trans-fected keratinocytes by DNA dot blot,RNA dot blot,hy-bridization in situ and immunohistochemistry.Western blot analysis showed a specific band exhibiting a molecular weight of15000which was in accordance with that of IL-2.The ac-tivity of IL-2in the culture supernatants was27.7U·mL-1 in2nd-passage cells and15U·mL-1 in4th-passage cells.CONCLUSION Keratinocytes have the potential availablity for gene stable expression and secreting active transgene products;This observations support the feasibility of ker-atinocytes as a target cell for gene transfection ......
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