File S1 - Efficient and Rapid C. elegans Transgenesis by Bombardment and Hygromycin B Selection

Contains: Figure S1. Survival of C. elegans on hygromycin B. Synchronized L1 larvae were added to seeded 6 cm NGM plates and immediately treated with the indicated hygromycin B concentrations. Plates were imaged 40 hours after treatment. a) At 300 worms per plate no treated worms reached the L4 stage even at the lowest hygromycin B concentration. b) At 3000 worms per plate some animals reached the L4 stage at the lowest antibiotic concentration, whereas at higher concentrations no animals reached the L4 stage. Figure S2. Gateway-based vectors constructed for the selection of hygromycin B resistant transgenics. a) Basic hygromycin B resistance vectors. b) New optimized hygromycin B resistance vectors. unc-54 3′UTR –3′ untranscribed downstream region of unc-54. Amp – ampicillin resistance gene. ori – origin of replication. attR4 & attR3– Gateway recombination sites. ccdB – toxicity gene. CAT - chloramphenicol acetyltransferase gene. MCS1 - KasI/NarI/SfoI, SpeI unique restriction sites. MCS2– KpnI, NheI, AvrII, AscI unique restriction sites. Prps-0_short – shorter upstream region (0.8 kb) of rps-0. HygR CeOPT – C. elegans optimized (codon optimization, introns) hygromycin B phosphotransferase gene. gpd-2/gpd-3 outron – CEOPX036 operon intergenic region (outron) between gpd-2 and gpd-3 genes. GFP – green fluorescent protein gene (optimized for C. elegans). mCherry – mCherry fluorescent protein gene (optimized for C. elegans). Figure S3. Hygromycin B independent transmission of integrated transgenes. Worms were judged to contain integrated transgenes using previously reported criteria: transmission frequency of 100%, no mosaicism in any worm [10]. An individual animal from an integrated transgenic strain was transferred to a single plate and a population grown until food was exhausted, at which time progeny were twice more transferred to fresh plates by chunking. The hygromycin B resistance gene and GFP were expressed under the control of the ubiquitous Prps-0 promoter (Prps-0::HygR::gpd-2/gpd-3::GFP::unc-54, bombardment 61, strains 61.4.3, 61.10.2; Prps-0_short::HygR, Prps-0::GFP::unc-54, bombardment 65, strain 65.2.1). Worms were imaged as described in the methods section. Exposure time: 700 ms. All of the strains were grown and propagated in the absence of hygromycin B. Table S1. Survival of C. elegans on hygromycin B. 300 (low density) or 3000 (high density) synchronized L1 larvae were plated on 6 cm plates and immediately treated with hygromycin B. The plates were then scored for the number of of L4 larvae (after ∼40 hours) and adult animals (after ∼60 and ∼90 hours). The experiment was repeated in triplicate. Table S2. Primers used for vector construction. Table S3. List of plasmids generated. Table S4. Detailed summary of all bombardments performed. Table S5. Optimization of bombardment conditions. The upper table shows the effect of rupture discs on transformation efficiency (rupture discs define the force at which gold beads are shot onto the worms). The lower table shows the effect of bead size in combination with different types of rupture discs. Table S6. Influence of DNA linearization on bombardment efficiency. To test the importance of DNA linearization on transformation efficiency, a single DNA mixture was prepared and then further processed in 3 different ways (non-linearized, linearized but not purified before coating, linearized and purified before coating). Purification was performed using a PCR purification kit (Qiagen). Supplementary Note. (DOC)