Selection of stably transfected cells expressing a high level of fetal muscle nicotinic receptors.
We had earlier found that the numbers of mouse muscle nicotinic receptors expressed on the surface of individual cells of a stably transfected clonal line of quail fibroblasts varied from cell to cell (Kopta and Steinbach: J Neurosci 14:3922-3933, 1994). We have now used repeated selective passages of these clonal cells to produce a population of cells which expresses a greater and more uniform number of surface receptors per cell. The increased level is stable over many cell divisions, and over many half-lives for the metabolic degradation of the surface receptors. Selection was performed by adhesion to a surface coated with a monoclonal antibody to a surface epitope on the muscle receptor, followed by expansion of the most tightly attached population of cells. Studies of the selected cells show that the surface receptors contain all four subunits of the muscle nicotinic receptor, and the functional properties of the receptors appear normal. The metabolic stability of the surface receptors is not altered, while the amount of mRNA for the subunits is increased in the selected population of cells. These observations indicate that the more likely reason for increased expression is a transcriptional effect, and that translational or posttranslational changes are unlikely.
Chen
Q
Q
Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Fletcher
G H
GH
Steinbach
J H
JH
D000818:Animals; D000911:Antibodies, Monoclonal; D002038:Bungarotoxins; D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006825:Hybridomas / Q000378:metabolism; D051379:Mice; D009132:Muscles / Q000166:cytology / Q000378:metabolism*; D011784:Quail; D012333:RNA, Messenger / Q000096:biosynthesis; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D000911:Antibodies, Monoclonal; D002038:Bungarotoxins; D012333:RNA, Messenger; D011978:Receptors, Nicotinic