DualTag knock-in does not affect the kinetics of transcriptional response upon stress. (A) Measurement of pre-mRNA abundance of target genes under stress in wild-type (without DualTag knock-in) by quantitative PCR. For icl-1, adh-1, acdh-1, and hach-1 group, worms at the L4 stage were fasted for various durations. For hsp-4 group, L4 stages worms were treated with or without tunicamycin for various durations. For hsp-16.41 group, worms synchronized to 1-day-old adults were collected immediately after heat shock at 30°C. Three biological replicates were quantified and shown. (B) Quantitative PCR measurement of pre-mRNA abundance of target genes under stress in animals with DualTag knock-in. All animals were similarly treated as in A. Three biological replicates were quantified and shown. (C) Schematic diagram showing the design of intronic probes to detect the nascent mRNAs of acdh-1 using smFISH. (D) Fluorescent images showing the abundance of the endogenous acdh-1 nascent mRNAs with or without fasting by smFISH in DualTag-knock-in animals. The probe against the bacterial DapB gene was used as a negative control. Green dots: MCP::GFP-positive acdh-1 RNA foci. Magenta dots: mRNAs tagged by smFISH probes. Nuclei were stained by DAPI (blue). For NC probe group, the boxed regions are magnified (2.3×). For acdh-1 intronic probe groups, the boxed regions are magnified (2×). Scale bars: 5 μm. (E) Comparison of acdh-1 nascent mRNAs detected by smFISH with or without fasting in animals with or without DualTag knock-in. The percentage of nuclei with RNA spots, number of active alleles per nucleus, and transcriptional output per nucleus in skin cells were quantified. The exact numbers of animals (left) or nuclei (middle and right) analyzed were indicated. All values are displayed as mean ± SEM. ns: not significant. ****P < 0.0001 (comparison between multiple groups with the same genotype: one-way ANOVA with the Tukey correction; comparison between two groups with different genotypes: two-tailed unpaired Student’s t test).
Figure 4.

DualTag knock-in does not affect the kinetics of transcriptional response upon stress. (A) Measurement of pre-mRNA abundance of target genes under stress in wild-type (without DualTag knock-in) by quantitative PCR. For icl-1, adh-1, acdh-1, and hach-1 group, worms at the L4 stage were fasted for various durations. For hsp-4 group, L4 stages worms were treated with or without tunicamycin for various durations. For hsp-16.41 group, worms synchronized to 1-day-old adults were collected immediately after heat shock at 30°C. Three biological replicates were quantified and shown. (B) Quantitative PCR measurement of pre-mRNA abundance of target genes under stress in animals with DualTag knock-in. All animals were similarly treated as in A. Three biological replicates were quantified and shown. (C) Schematic diagram showing the design of intronic probes to detect the nascent mRNAs of acdh-1 using smFISH. (D) Fluorescent images showing the abundance of the endogenous acdh-1 nascent mRNAs with or without fasting by smFISH in DualTag-knock-in animals. The probe against the bacterial DapB gene was used as a negative control. Green dots: MCP::GFP-positive acdh-1 RNA foci. Magenta dots: mRNAs tagged by smFISH probes. Nuclei were stained by DAPI (blue). For NC probe group, the boxed regions are magnified (2.3×). For acdh-1 intronic probe groups, the boxed regions are magnified (2×). Scale bars: 5 μm. (E) Comparison of acdh-1 nascent mRNAs detected by smFISH with or without fasting in animals with or without DualTag knock-in. The percentage of nuclei with RNA spots, number of active alleles per nucleus, and transcriptional output per nucleus in skin cells were quantified. The exact numbers of animals (left) or nuclei (middle and right) analyzed were indicated. All values are displayed as mean ± SEM. ns: not significant. ****P < 0.0001 (comparison between multiple groups with the same genotype: one-way ANOVA with the Tukey correction; comparison between two groups with different genotypes: two-tailed unpaired Student’s t test).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal