Pulser xcell

In attempts to generate the M. capsulatus Texas H156DRAFT_2759–2757 mutant, a linear DNA fragment was PCR-amplified from a plasmid containing a kanamycin resistance gene flanked by 1 kb M. capsulatus chromosomal sequence upstream of H156DRAFT_2759 and 1 kb downstream of H156DRAFT_2757. Electrocompetent M. capsulatus cells were prepared via the following protocol: a 300 mL culture was grown to mid-exponential phase, washed three times in 5% cold sterile glycerol and resuspended in 300 µL cold sterile 10% glycerol. The electrocompetent cells were mixed with 2.5 µL of the linear DNA fragment (1000 ng total) and shocked using various electroporation settings (Bacteria 1,2,3, and 5) on the Bio-Rad Pulser Xcell and the Ec1 setting on the Bio-Rad Micropulser [parameters were a combination of different cuvette gaps (0.1 cm or 0.2 cm), voltages (1.8–3.0 kV), resistances (200–400 Ω)]. Electroporated cells were grown up for 24 hr in 500 µL of media made with nitrate mineral salts (NMS) and methane; following this, the cells were plated on NMS-agar plates supplemented with kanamycin. No colonies were ever observed, suggesting that depletion of these transporters is toxic to the cells.

Knockdown of RNAs encoding SMDR2 (NCBI Acc. #L26287), SmMRP1 (NCBI Acc. #GU967672), ABCA4 (Smp_056290), ABCB6 (Smp_134890), and MRP7/ABCC10 (Smp_147250) was as described [47] (link), [81] (link). Briefly, following an overnight incubation in schistosome medium, adult worms (5 males plus 5 females) were placed in a 0.4 cm electroporation cuvette (USA Scientific, Ocala, FL) containing 50 µl siPORT (Life Technologies, Grand Island, NY) plus 3 µg of each of the siRNAs (IDT, Coralville, IA), either singly or in combination, targeting SMDR2, SmMRP1, MRP7, ABCA4, and ABCB6, or up to 15 µg luciferase siRNA (Life Technologies, Grand Island, NY; 3 µg per experimental siRNA used). The luciferase siRNA used for our control shows no significant similarity to any sequences from the S. mansoni gene database. siRNAs against the S. mansoni transporters were designed using the IDT SciTools RNAi Design server; sequences and targets are listed in Table 1. Worms were electroporated in this solution with a 20 ms square-wave pulse of 125 volts (BioRad Pulser XCell). Following electroporation, worms were incubated in schistosome medium for 2 days. They were then sorted into 2–3 males/female pairs per well in a 12-well plate, in which they were subsequently incubated in schistosome medium with carrier alone, or in medium plus PZQ (800 nM), as described above, and subsequently analyzed for motility.

Primary rat astrocytes were transfected with a plasmid encoding green fluorescent protein (GFP)-for the control group, or Nef for the experimental group (p96AM651 NIH AIDS Reference Research and Reagent Program, Cat. 8677, donated by Drs. Yingying Li, Feng Gao, and Beatrice H. Hahn). Transfections were performed with a Pulser Xcell (Bio-Rad, Hercules, CA), using 5 μg endotoxin-free DNA plasmid per 1.6 x 10⁶ cells, and pulsed with 250 V for 35 ms. After transfections, cells were resuspended in sterile artificial cerebrospinal fluid (ACSF) at a final concentration of 100,000 cells per 0.5 μL of ACSF.

A total of 1×10⁷ E14tg2a cells were electroporated (single pulse-250 V-path length 0.4 cm-500μF) with 10 μg of ScaI linearized CEC (chromosome elimination cassette) plasmid vector kindly supplied by T. Tada (Institute for Frontier Medical Sciences, Kyoto University, Japan) using a Pulser XCell (BioRad). Drug selection (500 ng/ml puromycin) was added after 48 h, and resistant clones were picked after 9 days.

The bacterial strains and plasmids used in this study are listed in Table S1. Xcc strains were routinely grown at 28 °C in rich medium NYG (5 g/L tryptone, 3 g/L yeast extract, and 20 g/L glycerol, pH 7), minimal and induction medium XVM2 (5 g/L glucose, 1 g/L sodium citrate, 2 g/L (NH₄)₂SO₄, 4 g/L K₂HPO₄, 6 g/L KH₂PO₄, and 0.2 g/L MgSO₄, pH 7.0) and XCM2 medium (2.36 g/L succinic acid, 0.15 g/L casamino acids, 1 g/L (NH₄)₂SO₄, 0.001 g/L MgSO₄, 10.5 g/L K₂HPO₄, and 8.35 g/L KH₂PO₄, pH 6.6). E. coli DH5α, cultured at 37 °C in Luria–Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7), was used to prepare all recombinant vectors needed. E. coli BL21(DE3) and E. coli M15 were used for protein expression. When required, the following concentrations of antibiotics were used: 100 μg/ml ampicillin, 50 μg/ml kanamycin, 100 μg/ml spectinomycin, and 25 μg/ml rifampicin. Electroporation was performed in a Bio‐Rad Pulser XCell at 18 kV/cm, 25 μF, and 200 Ω. All other general molecular biology operations were carried out according to standard molecular cloning protocols.