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

We have isolated genetic variants of the C2 muscle cell line that are defective in expressing the acetylcholine receptor (AcChoR). Because the AcChoR is expressed only after C2 myoblasts have fused to form myotubes, we employed a replica technique to detect the variants. This technique yields two copies of each clone, one of which can be used for screening and the other, as a source of dividing cells. In a screening of about 10,000 clones derived from mutagenized cells, we found 2 that 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 were found that failed to fuse and had reduced levels of both AcChoR and acetylcholinesterase. Though we relied on 125I-labeled alpha-bungarotoxin to distinguish wild-type from deficient clones, we found that an antiserum to the AcChoR, followed by a biotinylated second antibody and a horseradish peroxidase-avidin complex, could also be used. Therefore, it should be possible to obtain muscle cell variants defective in the expression of a variety of proteins for which specific antibodies are available.