Construction and expression of an adenosine deaminase::lacZ fusion gene
article
A eukaryotic expression vector was constructed in which the coding nucleotide sequences (ADA) of human adenosine deaminase (ADA) were fused in frame with the coding sequences of the bacterial gene lacZ encoding β-galactosidase (βGal). This ADA::lacZ fusion gene was anticipated to encode a hybrid protein that has retained the biological functions of both proteins. Transfection of mammalian cells with the fusion gene resulted in the synthesis of both ADA and βGal. Cells expressing the gene could therefore be detected with the histochemical staining procedure that relies on the conversion of the indicator, XGal, by βGal. In addition, the transfected cells ould be sorted on a fluorescence-activated cell sorter with the use of a vital staining procedure described for the selection of βGal-producing cells. Cell lines that harbored the fusion gene were tested for ADA overexpression by exposing them to the cytotoxic adenosine analog 9-β-D-xylofuranosyl adenine (Xyl-A), in the presence of the ADA inhibitor deoxycoformycin (dCF). Resistance to Xyl-A/dCF was observed in the lines carrying ADA::lacZ and moreover, the fraction of cells that survived a stringent selection for ADA overexpression also exhibited significantly increased levels of βGal, which confirmed the direct linkage between ADA and lacZ expression. The use of this and other fusion genes might be useful in the development of gene-therapy protocols where they could help to meet the demand for versatile methods to detect and select cells with newly introduced genes.
Chemicals/CAS: Adenosine Deaminase, EC 3.5.4.4; beta-Galactosidase, EC 3.2.1.23; Plasmids; Recombinant Fusion Proteins
Chemicals/CAS: Adenosine Deaminase, EC 3.5.4.4; beta-Galactosidase, EC 3.2.1.23; Plasmids; Recombinant Fusion Proteins
Topics
β-galactosidasefusion proteinhuman gene therapyrecombinant DNAadenosine deaminasebeta galactosidasearticlegene expressiongene fusiongene therapyhumanhuman cellnonhumantechniqueAdenosine DeaminaseAnimalbeta-GalactosidaseCell LineCloning, MolecularGene TherapyGenes, BacterialGenes, StructuralHumanPlasmidsRecombinant Fusion ProteinsRestriction MappingTransfection
TNO Identifier
231513
ISSN
03781119
Source
Gene, 98(2), pp. 283-287.
Pages
283-287
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