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WBPicture0000015226 Description: Figure 1. Functional Characterization of Germline-Specific Promoters in C elegans:(A) Properties of selected candidate promoters and summary of results. RNA-seq data from Serizay et al. (2020) is reported in Transcript Per Million (TPM). We calculated the relative germline expression as a fraction of expression in all major tissues (germline, neurons, intestines, hypodermis, and muscles). We selected promoter regions from the start codon of the candidate gene to the start or stop codon of the adjacent protein-coding gene. Homopolymer stretches of 10 bp or more and BsaI type IIS restriction sites in the selected promoter regions were modified by the introduction of base substitutions to facilitate synthesis and Golden Gate assembly. (B) Expression patterns of the ubiquitous eef-1A.1p (alternative name eft-3p) and germline mex-5p controls. (C-E) We classified germline-specific candidate promoters by careful visual inspection for somatic expression on a fluorescence microscope at high magnification (40x, oil objective). Due to very high GFP expression in the germline, somatic expression is not easily visible in all images. (C) GFP expression patterns of promoters that showed both somatic and germline fluorescence: his-61p, his-64p, hil-4p, rla-0p, rpl- 7Ap, and Y37E3.8p. (D) Promoters with no detectable GFP expression: puf-11p, F23A7.8p, and clec-87p. (E) Promoters with germline-specific expression: klp-19p, his-68p, W05F2.3p, spn-4p, pos-1p, Y75B12B.1p, puf-5p, mei-2p, and mesp-1p. Images were taken using a 20x air objective. Scale bars = 20 μm. (F) Top: Visual quantification of germline-specific GFP expression by scoring transgenic animals (blinded to genotype) from 0 (no expression) to 4 (high expression) on a fluorescence dissection microscope. Neg. Ctrl. refers to non-transgenic N2 animals. Bars indicate the mean, and error bars indicate the SEM. Bottom: Expression measurements using COPAS flow cytometry (Neg. Ctrl. (N = 64), mex-5p (N = 179), klp-19p (N = 134), his-68p (N = 156), W05F2.3p (N = 195), spn-4p (N = 160), pos-1p (N = 75), mei-2p (N = 238), Y75B12B.1p (N = 103), puf-5p (N = 104), mesp-1p (N = 91), puf-11p (N = 109), F23A7.8p (N = 54), clec-87p (N = 75)). Values plotted are relative to the mean of Neg. Ctrl. (non-transgenic N2 animals). Lines indicate the mean, and error bars indicate the SEM.
  Name: 4f091662fc7a7f06a70c85c516eed411.jpg
  Depict: Expr_pattern (15)
    Anatomy: WBbt:0005784
      WBbt:0008378
  Acknowledgment: Template: Reprinted from <Journal_URL>
    Publication_year: 2023
    Article_URL: DOI id 10.17912/micropub.biology.000843
    Journal_URL: microPublicationBiology
    Publisher_URL: Micropublication
  Reference: WBPaper00065569