Intron-embedded-MS2 has a smaller impact on gene expression compared to the insertion of MS2 into the UTR. (A) Cartoons showing different strategies to insert 24 copies of MS2 stem-loops for RNA imaging. (B) Confocal images showing mCherry protein expression in different knock-in strains. Noted that 24xMS2 were inserted into one of the introns of the mCherry or upstream of 3′UTR, respectively. mCherry insertions were used as controls. For all groups, worms at the fourth-larval stage (L4) were imaged and quantified. Bright-field images are scaled to 0.4× and shown at the top right in each image. Scale bars: 100 μm. (C) Relative protein expression levels of the strains showed in B were quantified. n represents the number of animals. (D) Relative mRNA abundance of target genes in animals showed in B measured by quantitative RT-PCR. Three biological replicates were quantified and shown. All values are displayed as mean ± SEM. ns: not significant. ****P < 0.0001 (one-way ANOVA with the Tukey correction).