Genetic engineering of hybridoma glutamine metabolism.
The murine hybridoma PQXB1/2 cannot be adapted to grow in culture media containing < 0.5 mM glutamine. Transformants selected following electroporation of PQXB1/2 cells with vectors containing a Chinese hamster glutamine synthetase (GS) cDNA under the control of the SV40 early promoter also failed to grow in the absence of glutamine in the culture medium. PQXB1/2 cells have, however, been transformed to glutamine independence following electroporation with a vector containing this glutamine synthetase cDNA under the control of the human cytomegalovirus immediate early promoter. In these cells, sufficient active glutamine synthetase was expressed from one vector per cell to enable growth in glutamine-free media. The specific activity of glutamine synthetase in two transformed cell lines producing parental levels of antibody was increased by 128 and 152%, respectively (0.57 and 0.63 mumol min-1 per 10(6) cells in transformants compared with parental levels of 0.25 mumol min-1 per 10(6) cells). This reprogramming of glutamine synthetase expression and glutamine metabolism is important for developing strategies to deal with ammonia toxicity and the production of cell lines with improved metabolic processes.
Bell
S L
SL
School of Biological Sciences, University of Surrey, Guildford, England.
Bebbington
C
C
Scott
M F
MF
Wardell
J N
JN
Spier
R E
RE
Bushell
M E
ME
Sanders
P G
PG
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D018076:DNA, Complementary / Q000235:genetics; D018274:Electroporation; D005818:Genetic Engineering; D005822:Genetic Vectors; D005974:Glutamate-Ammonia Ligase / Q000235:genetics; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D014162:Transfection
D018076:DNA, Complementary; D005973:Glutamine; D005974:Glutamate-Ammonia Ligase