Gene pulser xcell with ce module

For the knockdown of adult Hydra genes in vivo, Hydra polyps were electroporated with siRNAs, as previously described (47 (link), 48 (link)). Briefly, three siRNAs for each gene were designed and synthesized with a TT overhang (Genepharma Biotech) and tested for in vivo efficiency by reverse transcription quantitative PCR (RT-qPCR). siRNAs specific for each gene were selected as follows: control siRNA labeled by 6-carboxy-fluorescein (FAM) or not, 5′-AGGUAGUGUAAUCGCCUUG-3′; siHyCaspA, 5′-CUCGGAAGUAGACGUUCAATT-3′; siHyCARD2, 5′-CGGGCCUCUAAGAUCUUUATT-3′; siHyGSDME, 5′-CACCAUGUUUCAGAUUUAATT-3′, and scrambled sequence was designed as control: scrHyCaspA, 5′-CUACGAGUAGAUGACUGCATT-3′; scrHyCARD2, 5′-CGCCGGUCUGAUCUUAAUATT-3′; siHyGSDME, 5′-CCAUGUGAUUCUAUUACAATT-3′. For subsequent electroporation, 20 polyps were chilled on ice and then placed in a 0.4 cm–gapped plastic cuvette containing siRNAs (3 μM) diluted in 200 μl of double-distilled water. One square wave pulse (250 V) was applied for 25 ms (Gene Pulser XCell with CE Module; Bio-Rad). After electroporation, polyps were immediately transferred into 10 ml of Hydra medium supplemented with 20% (v/v) hyperosmotic dissociation medium to recover overnight at 18°C. Subsequently, the medium was changed with custom Hydra medium.

Expression plasmid containing the phr A gene was transformed into F. diplosiphon B481-WT according to parameters described by Tabatabai et al.^{14 (link)} Competent cells (40 μL) were mixed with
ligated purified plasmid DNA and electroporated using a GenePulser
Xcell with CE module (Bio-Rad) at 200 Ω resistance, 1.0 kV,
and 25 μF capacitance. After incubation on ice for 20 min, the
transformant was grown in BG11/HEPES liquid medium for 16 h and plated
on LB agar containing 80 mg L^–1 ampicillin. To verify
the insertion of the phr A gene, PCR was performed
using gene-specific primers as mentioned in Section 2.3, and products were visualized on a 1.5%
agarose gel.