An attempt to add biological functions by genetic engineering in order to produce high-performance bioreactor cells for hybrid artificial liver: transfection of glutamine synthetase into Chinese hamster ovary (CHO) cell.
In the course of immortalization, hepatocyte cell lines lose their original differentiated functions, such as ammonia removal and urea formation, drug metabolism, serum protein synthesis, etc. (Enosawa et al., Cell Transplant. 5:S39-S40; 1996). With the aim of adding lost or deficient functions and producing cell lines for the bioreactor of a hybrid artificial liver, rat glutamine synthetase (GS) gene was transfected into Chinese hamster ovary (CHO) cells, because it is able to lower the ammonia level. The GS gene-inserted pSV2 plasmid was transfected into the CHO-K1 line by electroporation. Transfected CHO (GS-CHO) cells were cultured in a glutamine-free medium containing ammonia, glutamic acid, and the GS inhibitor methionine sulfoximine (MSX). The MSX concentration was increased stepwise from 25 mumol/L to 1600 mumol/L to amplify the GS gene. In several GS-CHO sublines resistant to 300-1600 mumol/L of MSX, the specific activities of GS were increased from 0.2 x 10(4) to 1.7-2.9 x 10(4) unit/10(6) cells. When the amplified GS-CHO cells were cultured in the ammonia-containing medium, a slow but steady decrease of the ammonia level was observed when the level was high. Finally, the prospect of genetically modulated cells for bioreactors in the hybrid artificial liver is discussed.
Enosawa
S
S
Department of Experimental Surgery and Bioengineering, National Children's Medical Research Center, Tokyo, Japan.
Suzuki
S
S
Fujino
M
M
Amemiya
H
H
Omasa
T
T
Urayama
S
S
Tanimura
N
N
Suga
K
K
D000641:Ammonia / Q000378:metabolism; D000818:Animals; D019149:Bioreactors; D016466:CHO Cells; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005818:Genetic Engineering / Q000379:methods*; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000235:genetics*; D019164:Liver, Artificial; D008717:Methionine Sulfoximine / Q000494:pharmacology; D051381:Rats; D014162:Transfection
D004791:Enzyme Inhibitors; D008717:Methionine Sulfoximine; D000641:Ammonia; D005974:Glutamate-Ammonia Ligase