Journal: G3: Genes|Genomes|Genetics

Article Title: Population scale nucleic acid delivery to Caenorhabditis elegans via electroporation

doi: 10.1093/g3journal/jkab123

Figure Lengend Snippet: Optimization of electroporation conditions for C. elegans viability. General pipeline (A) of the electroporation procedure starts with the preparation of L1 synchronized worms (∼250), which are then mixed with electroporation buffer 80% in chilled cuvettes. After electroporation, worms are washed with 1 mL of M9 buffer and collected by centrifugation at 500 rcf for 2 min, then transferred to E. coli OP50 seeded plates to grow at 20° for 48 h. Animal survival rates (B) and body lengths (C) varied based on the electroporation conditions applied. The evaluation was performed using N2 animals for each pair of electroporation parameters with an electroporation pulse duration ranging from 5 to 25 ms and voltage ranging from 100 to 400 V. For (b), data were normalized to the initial calculated input number of worms (number of L1 worms was microscopically counted in 5 μl of the stock suspension before distribution between the experimental tubes). Animals placed in electroporation buffer without electric discharge were used as “untreated” controls. Worm survival rates: mean ± SD (standard deviation)% of two independent experiments. Body length measurements: here and on all other figures, if not stated otherwise, red lines indicate means, blue boxes show 25th and 75th percentiles, whiskers show the data distribution range. * P -values < 0.05 were considered statistically significant (ANOVA test with Bonferroni correction). Representative images (D) of worm populations exposed to electric discharges of different voltages (10 ms pulses) demonstrate the pronounced effect of the electroporation procedure on animal viability. Scale bar = 500 μm.

Article Snippet: Then 5 μl of worms were mixed with 40 μl of electroporation buffer (Gene Pulser Electroporation buffer, Biorad, Cat. No. 1652676) in 1.5 mL tubes, and allowed to incubate on ice for 5 min. An aliquot of 5 μl of purified dsRNA (10 μg/μl) was added to the worms just before the electroporation, mixed by pipetting, and transferred to 0.2 cm pre-chilled electroporation cuvettes (Biorad, Cat. No. 1652082).

Techniques: Electroporation, Centrifugation, Suspension, Standard Deviation

Journal: G3: Genes|Genomes|Genetics

Article Title: Population scale nucleic acid delivery to Caenorhabditis elegans via electroporation

doi: 10.1093/g3journal/jkab123

Figure Lengend Snippet: Identification of electroporation conditions for efficient delivery of dsRNA in C. elegans . To evaluate the effectiveness of nucleic acid delivery into animals, we used highly sensitive RNAi-mediated silencing of a GFP transgene following electroporation of dsRNA. (A) Synchronized L1 populations of BIG0107 [sid-2(gk505) III; Is(sur-5: gfp) I] worms (∼250) were electroporated with gfp -dsRNA of 1 μg/μl using favorable electroporation conditions. Animals placed in electroporation buffer without dsRNA or electric discharge were used as “untreated” controls. For each condition, GFP fluorescence intensity of worms ( n = 50) was measured in arbitrary units (a.u.). Asterisk (*) indicates groups where significant gfp silencing compared with the untreated control was observed ( P -value < 0.05, ANOVA test with Bonferroni correction). Results of body length comparison between worms electroporated at different conditions and untreated worms showed no significant differences (see Supplementary Figure S1A). (B) Three phenotypic categories of animals were scored in each condition group, including worms with “No silencing”, “Partial gfp silencing,” and “Complete gfp silencing” (all but neuronal cells). n = number of worms scored. Representative images of worms from each category are shown, scale bar = 100 μm. The electroporation parameters of 300 V 10 ms with the highest percentage in “Complete gfp silencing” category (59%) were chosen as the most efficient. (C) Populations of BIG0107 animals at L1, L4, and YA stages were electroporated with gfp -dsRNA of 1 μg/μl using electroporation conditions of 300 V 10 ms. Levels of GFP fluorescence were measured in 48 h after electroporation and compared to the control worms including untreated animals [electroporation (−)/dsRNA (−)], electroporated animals without dsRNA [electroporation (+)/dsRNA (−)], and animals incubated with dsRNA [electroporation (−)/dsRNA (+)] ( P -values are noted, ANOVA test with Bonferroni correction). No significant differences in worm body lengths were observed (see Supplementary Figure S1, B–D).

Article Snippet: Then 5 μl of worms were mixed with 40 μl of electroporation buffer (Gene Pulser Electroporation buffer, Biorad, Cat. No. 1652676) in 1.5 mL tubes, and allowed to incubate on ice for 5 min. An aliquot of 5 μl of purified dsRNA (10 μg/μl) was added to the worms just before the electroporation, mixed by pipetting, and transferred to 0.2 cm pre-chilled electroporation cuvettes (Biorad, Cat. No. 1652082).

Techniques: Electroporation, Fluorescence, Control, Comparison, Incubation

Journal: G3: Genes|Genomes|Genetics

Article Title: Population scale nucleic acid delivery to Caenorhabditis elegans via electroporation

doi: 10.1093/g3journal/jkab123

Figure Lengend Snippet: Evaluation of tissue distribution of RNAi silencing in electroporated animals. (A, B) Representative images of BIG0106 [sid-1(qt9) V; Is(sur-5::gfp) I] and BIG0107 [sid-2(gk505) III; Is(sur-5::gfp) I] worms were taken 48 h after the electroporation of L1 worm populations with gfp -dsRNA of 1 μg/μl using 300 V 10 ms conditions. Images of “untreated” control animals [electroporation (−)/dsRNA (−)] are presented for comparison. Scale bar = 100 μm. Additional images of these worms presented in three digital focal planes can be found in supplementary information (Supplementary Figures S3 and S4). (C) Levels of GFP fluorescence in [sid-1(qt9) V; Is(sur-5::gfp) I] worm strains ( n = 15) after electroporation were compared to the controls worms ( P -values are noted, ANOVA test with Bonferroni correction). No significant differences in body lengths between electroporated and control worms were observed (Supplementary Figure S2). (D) Schematic of the presumed routes of dsRNA transport in sid-1 (+)/ sid-2 (−) animals highlights hypodermal entry as a primary site of initial dsRNA delivery by electroporation, followed by spread to other tissues in a SID-1-dependent manner.

Article Snippet: Then 5 μl of worms were mixed with 40 μl of electroporation buffer (Gene Pulser Electroporation buffer, Biorad, Cat. No. 1652676) in 1.5 mL tubes, and allowed to incubate on ice for 5 min. An aliquot of 5 μl of purified dsRNA (10 μg/μl) was added to the worms just before the electroporation, mixed by pipetting, and transferred to 0.2 cm pre-chilled electroporation cuvettes (Biorad, Cat. No. 1652082).

Techniques: Electroporation, Control, Comparison, Fluorescence

Journal: G3: Genes|Genomes|Genetics

Article Title: Population scale nucleic acid delivery to Caenorhabditis elegans via electroporation

doi: 10.1093/g3journal/jkab123

Figure Lengend Snippet: Efficiency of electroporation-driven gene silencing of endogenous genes is dose dependent. In order to test the effectiveness in nontransgenic animals, we targeted endogenous hypodermally expressed genes with robust RNAi phenotypes, nhr-23 [larval arrest, (A–C)] and dpy-13 [shortened body size, (D–F)]. (A) Impact of electroporation of nhr-23 -dsRNA on the development of sid-2(gk505) worms treated at L1 stage and imaged after 48 h. Red lines indicate means, blue boxes show 25th and 75th percentiles, whiskers show the data distribution range. (B) Proportion of animals scored as having either “normal” or “delayed” development after electroporation. (C) Representative image of worms show the nhr-23 silencing effect at 1 μg/μl of dsRNA (right image), when compared to untreated worms (left image). Scale bar = 500 μm. (D) Impact of electroporation of dpy-13 -dsRNA on body size of sid-2(gk505) worms treated at L1 stage and imaged after 72 h. Red lines indicate means, blue boxes show 25th and 75th percentiles, whiskers show the data distribution range. (E) Proportion of animals scored as “normal” or “dumpy” after electroporation. (F) Representative images of worms demonstrate the dpy-13 silencing at 1 μg/μl of dsRNA (right image) in comparison with untreated control worms (left image). Scale bar = 500 μm. Asterisk (*) indicates groups with significant gene silencing compared to the untreated control (ANOVA test with Bonferroni correction).

Article Snippet: Then 5 μl of worms were mixed with 40 μl of electroporation buffer (Gene Pulser Electroporation buffer, Biorad, Cat. No. 1652676) in 1.5 mL tubes, and allowed to incubate on ice for 5 min. An aliquot of 5 μl of purified dsRNA (10 μg/μl) was added to the worms just before the electroporation, mixed by pipetting, and transferred to 0.2 cm pre-chilled electroporation cuvettes (Biorad, Cat. No. 1652082).

Techniques: Electroporation, Comparison, Control

Journal: G3: Genes|Genomes|Genetics

Article Title: Population scale nucleic acid delivery to Caenorhabditis elegans via electroporation

doi: 10.1093/g3journal/jkab123

Figure Lengend Snippet: Evaluation of electroporation for delivery to the animal germline and progeny. To further test the utility of this approach, we sought to identify whether we could, first, stimulate RNAi knockdown of endogenous gene pos-1 expressed in germline with robust phenotype (embryonic lethality) and, second, deliver guide RNA (gRNA) for CRISPR/Cas9-mediated genome editing of the endogenous dpy-10 gene. (A) Schematic of pos-1 -dsRNA delivery to L4 worms by electroporation (300 V for 10 ms, 1 μg/μl of dsRNA in electroporation buffer with final volume of 50 μl) followed by phenotypic analysis of progeny. Animals were allowed to lay eggs for 24 h, removed from the lawn, and the proportion of hatched progeny was determined after 48 h. (B) Representative images of effective electroporation-mediated delivery of pos-1 -dsRNA to sid-2(gk505) animals. Scale bar = 500 μm. Proportions of hatched/unhatched embryos from individual worms after pos-1 -dsRNA electroporation compared to control animals demonstrated in Supplementary Figure S6. (C) Schematic of delivery of dpy-10 gRNA to Young Adult (YA) animals by electroporation (300 V for 10 ms, 1 μg/μl of RNA in electroporation buffer with final volume of 50 μl) followed by phenotypic screening of progeny for evidence of genome editing (Dumpy or Roller). (D) Representative images of successful electroporation of dpy-10 gRNA in EG9888 animals that resulted in F1 progeny with visible Rol and Dpy phenotypes. Scale bar = 300 μm. (E) Illumina sequencing-based confirmation of Cas9-mediated mutations.

Article Snippet: Then 5 μl of worms were mixed with 40 μl of electroporation buffer (Gene Pulser Electroporation buffer, Biorad, Cat. No. 1652676) in 1.5 mL tubes, and allowed to incubate on ice for 5 min. An aliquot of 5 μl of purified dsRNA (10 μg/μl) was added to the worms just before the electroporation, mixed by pipetting, and transferred to 0.2 cm pre-chilled electroporation cuvettes (Biorad, Cat. No. 1652082).

Techniques: Electroporation, Knockdown, CRISPR, Control, Illumina Sequencing