Selection for phenylalanine hydroxylase activity in cells transformed with recombinant retroviruses.
Cells deficient in phenylalanine hydroxylase (PAH) are tyrosine auxotrophs and will not survive in tyrosine-free media. PAH activity can be constituted in cultured cells by infection with recombinant retroviruses carrying a human PAH cDNA. Mouse hepatoma cells transformed with recombinant PAH will grow in tyrosine-free media since these cells constitutively synthesize the cofactor tetrahydrobiopterin which is essential for PAH activity. NIH3T3 cells transformed with the PAH cDNA express the PAH apoenzyme, but this enzyme is inactive in vivo since these cells do not synthesize biopterin. We describe a method of selection for PAH in the fibroblast-like NIH3T3 cells involving tyrosine-free media supplemented with biopterin, reducing agents, and antioxidants. Cells transformed with the recombinant PAH gene exhibit PAH activity in culture and will grow in the biopterin-supplemented tyrosine-free media. Metabolic selection for PAH activity provides a new selectable marker for gene transfer experiments. This method is shown to be useful in the production of high titers of recombinant retroviruses carrying PAH and provides a model for experiments in somatic gene therapy of phenylketonuria.
Ledley
F D
FD
Hahn
T
T
Woo
S L
SL
D000818:Animals; D002460:Cell Line; D003470:Culture Media; D005819:Genetic Markers; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D009154:Mutation; D010651:Phenylalanine Hydroxylase / Q000172:deficiency / Q000235:genetics*; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection
D003470:Culture Media; D005819:Genetic Markers; D010651:Phenylalanine Hydroxylase