Heritable and Lineage-Specific Gene Knockdown in Zebrafish Embryo
Figure 2
Knockdown of EGFP sensors by mir-shRNA in vivo.
(A) Diagram of miRNA-based shRNAEGFP-ORF (mir-shRNAEGFP-ORF). The stem-loop region of miR-30e precursor was replaced with chemically synthesized shRNAEGFP-ORF oligonucleotides containing the same sequence as the miR-30e stem-loop, except that the miR-30e hairpin stem was changed to the sequence that was complementary to the open reading frame (ORF) of EGFP transcript. (B) Northern blot analysis of 12 and 24 hpf embryos injected with in vitro synthesized mir-shRNAEGFP-ORF mRNAs. The U6 promoter-driven expression of shRNAEGFP-ORF in 293T cells was used as a positive control (left lanes). (C) Diagram of various sensors containing one or two copies of binding sequence for mir-shRNAEGFP-ORF within the ORF or the 3′UTR. The 22-bp long binding sequence was inserted into the 3′UTR-SV40 of either EGFP or DsRed gene. (D) Individual capped sensor mRNAs was co-injected with either miR-30e precursor control or mir-shRNAEGFP-ORF. (E–H) Detection of EGFP and DsRed fluorescence in 24 hpf embryos injected with various sensor mRNAs. Red fluorescence was used as an injection control in E, F and G, and green fluorescence as injection control in H. (I) Western blot analysis of 24 hpf embryos shown in panels E–G. The β-actin was used as a loading control.