Fig. 1.

Confirmation of transgenics. (A) Sanger traces confirming that the mutations are present in the transgenes integrated into the C. elegans strains. Mutation sites are indicated by red boxes. (B) Confirmation of gene expression by RT-PCR (primers seqhVMATf1 and seqhVMATr1). RT negative controls contained no reverse transcriptase. P237H (NZ), P387L (SA), WT. (C) Prior to Sanger sequencing, C. elegans were genotyped by PCR. Each C. elegans required 2 wells for assessment: the first well contains a positive control (SSU18A+SSU26R ∼950 bp) and a test for the transgene (primers unc5402 and seqSNB01, 2657 bp). Primers NM3884 and NM3880, which flank the integration site, were used to determine insertion of the construct. The second well for each sample only show a product (2945 bp) if one chromosome does not have the construct inserted, and no band if the C. elegans is homozygous. The top series of bands are all from C. elegans of a single parent, deemed heterozygous, as all possible genotype combinations are represented. The lower bands are from a homozygous parent, as all C. elegans show presence of the transgene, and a copy on each chromosome. Primer sequences are available in Table S3. TG, transgene.