eft 3 p cas9 sgrna expression vector  (Addgene inc)

Journal: Current biology : CB

Article Title: Ectopic germ cells can induce niche-like enwrapment by neighboring body wall muscle

doi: 10.1016/j.cub.2019.01.056

Figure Lengend Snippet: (A) Maximum intensity z-projections through 5 μm of the DTC central plexus (top left, lag-2p::GFP::CAAX) and body wall muscle cell membrane (bottom left, myo-3p::GFP:: CAAX). Germ cells (second column) are enwrapped both in the endogenous niche (top) and ectopically by muscle (bottom). Boxes in overlay (third) show areas of enlargement (right) in which both DTC (top) and muscle (bottom) have thin, enwrapping projections that surround germ cells. (B) Control (top) animal expressing lag-2p::GFP::CAAX (left) in the DTC (arrow) and muscle (arrowhead) after epi-1 RNAi have a robust germ cell population (center, mex-5p::H2B::mCherry). DTC-ablated animals (bottom) had few germ cells 48 h post-ablation compared to controls. Single confocal z-slices are shown. Full projection of the control animal also appears in Figure S5. (C) The DTC niche (left, lag-2p::myr::tdTomato) expresses the CRISPR/Cas9-tagged Notch ligand LAG-2::mNeonGreen (center), which localizes in a punctate pattern in the DTC membrane in one-day adults. Maximum intensity z-projections through 2.5 μm of confocal slices are shown. (D) Body wall muscles (left, myo-3p::GFP::CAAX) do not express LAG-2::mNeonGreen (center, punctate DTC expression visible), overlay (right). Maximum intensity z-projections through 5 μm of confocal slices are shown. Scale bars are 10 μm.

Article Snippet: eft-3 p::Cas9+sgRNA expression vector , [ 33 ] , pDD162, Addgene plasmid #47549.

Techniques: Expressing, CRISPR

Journal: Current biology : CB

Article Title: Ectopic germ cells can induce niche-like enwrapment by neighboring body wall muscle

doi: 10.1016/j.cub.2019.01.056

Figure Lengend Snippet: (A) A control DTC (upper left, L4440 vector) has more intercalating processes than the DTC of an animal with hmr-1(RNAi) in the sax-7(eq1) background (lower left), compared to mild defects observed in sax-7(eq1) animals (lower right) and hmr-1(RNAi) alone (upper right). Intercalating processes (arrowheads) are quantified in (B). Maximum intensity core z-projections through 10 μm of confocal slices are shown. (B) Box plots quantifying intercalating processes for genotypes in (A). *p < 0.05, **p < 0.005, ***p < 0.0005, n ≥ 24 animals for each group, ANOVA followed by Tukey-Kramer HSD test. (C) Animals treated with epi-1 RNAi to induce rupture (left, ectopic germ cells visible in DIC image) expressing CRISPR/Cas9-tagged HMR-1::GFP (center) and muscle membrane marker (right, myo-3p::mCherry::PLCδPH). Control (top) shows full enwrapment of all germ cells along the dorsal body wall (yellow asterisks). RNAi against hmr-1 (bottom) before epi-1 RNAi causes reduced HMR-1::GFP (center, except in uterus, arrowhead), and reduced enwrapment of germ cells (germ cells that are not enwrapped marked by blue X). RNAi treated animals also lack body-crossing muscle (white dashed line) protrusions. RNAi against hmr-1 not only decreased the number of ectopic germ cells enwrapped, but also the amount of muscle cell membrane forming muscle protrusions, which explains why fluorescence in the right RNAi panel is dimmer than the control. (D) The number of animals with full enwrapment is decreased after hmr-1 RNAi treatment. Fisher’s exact text, two-tailed ***p < 0.0005. Wide field DIC and fluorescent images are shown. The candidate gene hmr-1 was identified with an RNAi screen, see Table S1. Scale bars are 10 μm.

Article Snippet: eft-3 p::Cas9+sgRNA expression vector , [ 33 ] , pDD162, Addgene plasmid #47549.

Techniques: Plasmid Preparation, Expressing, CRISPR, Marker, Fluorescence, Two Tailed Test