Use of a replica technique to isolate muscle cell lines defective in expressing the acetylcholine receptor.

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Abstract
Genetic variants of the [mouse] C2 muscle cell line that are defective in expressing the acetylcholine receptor (AcChoR) were isolated. Because the AcChoR is expressed only after C2 myoblasts have fused to form myotubes, a replica technique was used to detect the variants. This technique yields 2 copies of each clone, 1 of which can be used for screening and the other, as a source of dividing cells. In a screening of .apprx. 10,000 clones derived from mutagenized cells, 2 fused normally and expressed normal levels of acetylcholinesterase but had reduced amounts of AcChoR on their surface. One of these also had a reduced level of intracellular AcChoR, but, in the other, the amount of intracellular AcChoR was 5-fold higher than normal. Several variants failed to fuse and had reduced levels of both AcChoR and acetylcholinesterase. Though 125I-labeled .alpha.-bungarotoxin was used to distinguish wild-type from deficient clones, an antiserum to the AcChoR, followed by a biotinylated 2nd antibody and a horseradish peroxidase-avidin complex, could also be used. It should be possible to obtain muscle cell variants defective in the expression of a variety of proteins for which specific antibodies are available.