Construction and expression of an adenosine deaminase::lacZ fusion gene.
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 beta-galactosidase (beta Gal). 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 beta Gal. Cells expressing the gene could therefore be detected with the histochemical staining procedure that relies on the conversion of the indicator, XGal, by beta Gal. In addition, the transfected cells could be sorted on a fluorescence-activated cell sorter with the use of a vital staining procedure described for the selection of beta Gal-producing cells. Cell lines that harbored the fusion gene were tested for ADA overexpression by exposing them to the cytotoxic adenosine analog 9-beta-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 beta Gal, 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.
Shen
Q
Q
Institute of Applied Radiobiology and Immunology-TNO, Rijswijk, The Netherlands.
van Beusechem
V W
VW
Einerhand
M P
MP
Hendrikx
P J
PJ
Valerio
D
D
D000243:Adenosine Deaminase / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D005796:Genes; D005798:Genes, Bacterial; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D000243:Adenosine Deaminase