Stable expression of transfected Torpedo acetylcholine receptor alpha subunits in mouse fibroblast L cells.
Torpedo californica electric organ cDNA libraries were constructed in lambda gt10 and lambda gt11. Four acetylcholine receptor (AcChoR) subunit cDNA clones were isolated and shown to contain the entire coding region for each of the subunits. When in vitro synthesized AcChoR mRNA was microinjected into Xenopus laevis oocytes, functional cell surface AcChoRs were expressed. A very simple and fast 22Na-uptake experiment was performed on batches of microinjected oocytes to identify oocytes that were expressing large quantities of functional cell surface AcChoRs for use in single-channel recordings. In addition to the transient expression system, DNA-mediated cotransformation is described, which is a method for stably introducing AcChoR cDNAs into the chromosomes of tissue culture cells. Because the AcChoR is composed of four different subunits, it is necessary to integrate four cDNAs into the chromosomes of the same cell before stable expression of a completely functional receptor complex can be established. We show that 80% of the cells that integrated the selectable marker gene into their chromosomes also integrated all four AcChoR cDNAs. When Torpedo alpha-subunit cDNA inserted into an appropriate expression vector was introduced into cells by transfection, alpha-subunit protein was synthesized that migrated on NaDodSO4/polyacrylamide gels with the same molecular mass as native Torpedo alpha subunits and expressed antigenic determinants similar to those of native Torpedo alpha subunits.
D000818:Animals; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D004557:Electric Organ / Q000032:analysis; D005347:Fibroblasts / Q000378:metabolism*; D005786:Gene Expression Regulation; D009865:Oocytes / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000235:genetics*; D014101:Torpedo; D014162:Transfection
D011950:Receptors, Cholinergic; D004247:DNA