Comparing engineered nuclear-localized reporter cassettes

Abstract

Recent single-cell transcriptome analysis has revealed a tremendous breadth and specificity of neuropeptide-encoding gene expression in the nervous system of C. elegans. To analyze the dynamics of neuropeptide gene expression, as well as to dissect the regulatory mechanism by which their expression is controlled, reporter genes remain an important tool. Using CRISPR/Cas9 genome-engineering, we generate here reporter alleles for 6 different neuropeptide encoding genes (3 flp genes, 1 nlp and 2 insulin genes). We find that different reporter cassettes result in different levels of reporter expression and recommend usage of an SL2::GFP::H2B or GFP::H2B::SL2 cassette.

Figure 1. Comparing engineered nuclear-localized reporter cassettes

Fig 1. (A) Schematic of reporter cassette comparison. (B) Comparison of the different reporter cassettes for neuropeptide nlp-51 . Analysis for nlp-51 GFP fluorescence in the RIP and AIM neurons is shown on the left, with representative images on the right. (C) Comparison of the different reporter cassettes for neuropeptide flp-20 . Analysis for flp-20 GFP fluorescence in the LUA, PVR, PVC, and PLM neurons is shown on the left, with representative images on the right. For B and C, *P<0.05, **P<0.01, ***P<0.001 posthoc t-test with Tukey multiple comparison correction. The black bars on the bottom right corner of microscope images represent 10 µm. (D) Summary of T2A::3xNLS::GFP vs. SL2::GFP::H2B comparisons across 6 different neuropeptide genes. Expression is determined using the reporter alleles in combination with NeuroPAL (Yemini et al., 2021), and the expression pattern of the SL2::GFP::H2B cassette more closely matches scRNA-seq data (Taylor et al., 2021).