Figure 4.

Luciferase activity in Ascaris embryos after biolistic introduction of RNA. Luciferase RNAs lyophilized onto gold particles (typically 5 μg RNA/mg gold) were introduced into 32- to 64-cell Ascaris embryos, and luciferase activity was measured in the embryos after incubation at 30°C. Relative luciferase light (RLU) units represent only ≈15–20% of the total activity in the embryos. (A) Structure of the in vitro-generated luciferase RNA. (B) Luciferase expression is RNA dependent. Embryos were assayed for luciferase activity 8 hr after RNA biolistics. Nuclease treatment of the RNA preceeded lyophilization of the RNA onto gold particle (n = 4 from two separate experiments). Maximum luciferase activity in a representative experiment was 236,423 ± 10,653 (n = 2), which represents ≈15% of the total embryo activity. No Nuclease Treatment, RNA introduced without additional treatment; DNase Treated, RNA treated with DNase I before introduction into embryos; Transcription Template, PCR generated template used for in vitro transcription of the luciferase RNA; RNA Assayed, equivalent amounts of RNA lyophilized onto beads directly assayed for luciferase activity. (C) Time course of luciferase activity in Ascaris embryos (n = 3). (D) Dose response for luciferase RNA expression in Ascaris embryos (n = 3). Varying amount of RNA (1.25–10 μg) were lyophilized onto gold particles (1 mg), and luciferase activity was measured 6 hr after biolistic introduction of the RNA.