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    188宝金博页面版: Comparison of HCMV IE and EF-1 Promoters for the Stable Expression of β-Subunit of Hexosaminidase in CHO Cell Lines

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    内容提示： Biochemical Genetics, Vol. 44, Nos. 3/4, April 2006 ( C ? 2006)DOI: 10.1007/s10528-006-9016-3NoteComparison of HCMV IE and EF-1α Promotersfor the Stable Expression of β-Subunit ofHexosaminidase in CHO Cell LinesIncilay Sinici, 1,3 Maryam Zarghooni, 2 Michael B. Tropak, 2Don J. Mahuran, 2 and H. Asuman¨Ozkara 1Received 11 May 2005—Final 20 October 2005Published online: 28 April 2006INTRODUCTIONThe ability to use in vitro mutagenesis and transfection techniques to generatestable cell lines that highly ex...

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    Biochemical Genetics, Vol. 44, Nos. 3/4, April 2006 ( C ? 2006)DOI: 10.1007/s10528-006-9016-3NoteComparison of HCMV IE and EF-1α Promotersfor the Stable Expression of β-Subunit ofHexosaminidase in CHO Cell LinesIncilay Sinici, 1,3 Maryam Zarghooni, 2 Michael B. Tropak, 2Don J. Mahuran, 2 and H. Asuman¨Ozkara 1Received 11 May 2005—Final 20 October 2005Published online: 28 April 2006INTRODUCTIONThe ability to use in vitro mutagenesis and transfection techniques to generatestable cell lines that highly express a mutant protein associated with a metabolicdisease has been an important technique in linking genotype to clinical pheno-type ( ¨ Ozkara and Sandhoff, 2003). One group of metabolic diseases is GM2gangliosidosis (AB-variant, Tay-Sachs and Sandhoff diseases). These diseases arecharacterized by the accumulation of GM2 ganglioside, primarily in the lyso-some of neuronal tissue, leading to progressive neurodegeneration. Tay-Sachsand Sandhoff diseases are caused by defects in the HEXA gene, which encodesthe α-subunit, and the HEXB gene, which encodes the β-subunit of heterodimericN-acetyl-β-hexosaminidase(Hex)(EC3.2.1.52),respectively(Graveletal.,2001;Mahuran, 1999). The AB-variant is associated with defects in the GM2 activatorprotein, a substrate-specif i c cofactor for Hex A. The second major Hex isozymein normal human tissue is Hex B, a homodimer of β-subunits.In this study, we introduced a cDNA encoding the β-subunit of human Hex(βcDNA) into three different eukaryotic expression vectors containing two differ-ent promoters, in order to establish which produces the highest expression levelsof Hex B activity and β-protein in permanently transfected CHO cells. Our resultsindicate that the type of promoter employed can be critical in the establishment ofhighly expressing cell lines.1 Department of Biochemistry, Faculty of Medicine, Hacettepe University, 06100 Ankara, Turkey.2 Research Institute, Hospital for Sick Children, Toronto, Canada.3 To whom correspondence should be addressed; e-mail: isinici@hacettepe.edu.tr.1730006-2928/06/0400-0173/1 C ? 2006 Springer Science+Business Media, Inc.

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