Simultaneous expression of multiple proteins under a single promoter in Caenorhabditis elegans via a versatile 2A-based toolkit

Abstract

Caenorhabditis elegans is a powerful in vivo model in which transgenesis is highly developed. However, while the analysis of biological phenomena often require the expression of more than one protein of interest, no reliable tool exists to ensure efficient concomitant and equivalent expression of more than two polypeptides from a single promoter. We report the use of viral 2A peptides, which trigger a "ribosomal-skip" or "STOP&GO" mechanism during translation, to express multiple proteins from a single vector in C. elegans. Although none of the viruses known to infect C. elegans contain 2A-like sequences, our results show that 2A peptides allow the production of separate functional proteins in all cell types and at all developmental stages tested in the worm. In addition, we constructed a toolkit including a 2A-based polycistronic plasmid and reagents to generate 2A-tagged fosmids. 2A peptides constitute an important tool to ensure the delivery of multiple polypeptides in specific cells, enabling several novel applications such as the reconstitution of multi-subunit complexes.

Keywords: 2A peptide; Caenorhabditis elegans; SL2; internal ribosomal entry site (IRES); stoichiometric delivery of multiple polypeptides.

Figure 1

Each 2A viral peptide allows the efficient production of two independent polypeptides in C. elegans. (A) Patterns of localization of GFP and HISTONE::mCherry in the rectal cells of L4 worms expressing col-34p::GFP::2A::histone::mCherry. GFP localizes within the whole cell whereas HISTONE-mCherry (HIST::mCherry) remains tightly restricted to the nucleus as expected if they are produced as two independent polypeptides, suggesting that all 2A peptides are correctly split up. For the mutant P2A* peptide, both markers are restricted to the nucleus as expected if the split-up activity is abolished. SL2, used as a positive control, triggers the production of two RNA species. In the merge column, the rectal cells B, F, U, K, and Y are labeled (capital letters); the nucleus (n.) and cytosol (c.) are labeled in all columns; the dotted line indicates the rectal slit; anterior is to the left and ventral to the bottom. (B) Quantification of the localization patterns observed for col-34p::GFP::2A::histone::mCherry in rectal cells of L1 worms; n, number of cells scored. (C) Western blot analysis showing the production of independent GFP and HISTONE-mCherry proteins. Red arrows on the left indicate GFP and HISTONE-mCherry bands detected at ∼30 and 60 kDa, respectively; dashed arrows on the right indicate noncleaved product. To obtain enough protein to perform the Western blot analyses, we used the myo-3 promoter, which drives expression in the 81 BWM cells of the worm; α-tubulin was used as a loading control; a SL2 trans-splicing line, resulting in the expression of two independent products, was used as a reference. Note that GFP migrates slightly slower in extracts from transgenic 2A lines as the majority of the 2A peptide remains fused to the C terminus of GFP. All transgenic lines obtained are listed in Table S3.

Figure 2

Each 2A viral peptide allows the efficient production of two independent polypeptides at different developmental stages. (A) Quantification at different developmental stages of the expected localization patterns (GFP in the whole cell, HISTONE::mCherry in the nucleus) observed in lines expressing myo-3p::GFP::E2A::histone::mCherry, myo-3p::GFP::P2A*::histone::mCherry, or myo-3p::GFP::SL2::histone::mCherry. (B) Patterns of localization of GFP and HISTONE::mCherry in muscle cells of myo-3p::GFP::F2A::histone::mCherry or myo-3p::GFP::P2A*::histone::mCherry transgenic worms at the indicated developmental stages. n, nucleus; c, cytosol. See Table S3 for information on the different transgenic lines obtained.

Figure 3

2A viral peptides allow the efficient production of two independent polypeptides in different tissues. (A) Localization pattern of GFP and HISTONE::mCherry in neuronal cells in transgenic lines expressing a F25B3.3p::GFP::T2A::histone::mCherry construct. GFP localizes within the whole cell whereas HISTONE-mCherry remains tightly restricted to the nucleus. n, nuclear; c, cytosol. The ventral midbody area of the worm is shown. The worm is oriented vertically with its tail to the bottom. (B) Western blot analysis showing the production of independent GFP and mCherry proteins from a T2A construct in neuronal cells. The GFP and HISTONE-mCherry products, detected at 30 and 60 kDa, respectively, are indicated by red arrows; dotted arrows indicate the expected size for an uncleaved product; α-tubulin was used as a loading control. (C) Localization pattern of GFP and HISTONE::mCherry in intestinal cells of embryos and L1 larvae in lines expressing a ugt-22p::GFP::T2A::histone::mCherry construct. GFP localizes within the whole cell whereas HISTONE-mCherry remains tightly restricted to the nucleus. n, nuclear; c, cytosol. The midbody area of L1 larvae oriented vertically with the tail to the bottom is observed. See Table S3 for information on the different transgenic lines obtained.

Figure 4

2A viral peptide technology allows simultaneous delivery of multiple independent and functional polypeptides in the worm. (A) Schematic view of the molecular construct encompassing five products fused in frame with F2A, T2A, E2A, and P2A. GFP is addressed to the whole cell, mCherry::PH (Pleckstrin Homology domain) is addressed to the cellular membrane, EMR-1::cerulean (Ce-emerin) is addressed to nuclear membrane (Lee et al. 2000), and HISTONE::mCherry is addressed to the nucleus. col-34p was used to drive expression in the rectal cells (Kagias et al. 2012). (B) Confocal imaging of rectal cells of early L1s expressing col-34p::GFP::F2A::mCherry-PH::T2A::sem-4A-HA::E2A::emr-1::cerulean::P2A::histone::mCherry. All the markers are addressed to the expected cellular compartments (see also Figure S5); the Y, B, and F rectal cells are shown; anterior is to the left and ventral to the bottom. (C) Quantification of the presence of all five markers at the correct compartment. We noted in one of the two lines (IS2008) that the fifth protein, HISTONE::mCherry, is not seen in 3.3% of the cells scored. n, total number of cells scored. (D) Western blot analysis showing the presence of SEM-4a::HA as one band at ∼100 kDa as expected (white arrow) in the two transgenic independent lines IS2008 and IS2070. (E) Rescuing efficiency of the sem-4(n1971) “no PDA” defect in the two independent lines IS2008 and IS2070 expressing col-34p::GFP::F2A::mCherry-PH::T2A::sem-4A-HA::E2A::emr-1::cerulean::P2A::histone::mCherry. Interestingly, the rescuing efficiency for each line correlated with the levels of expression of SEM-4a-HA detected by Western Blot. For each experiment, nontransgenic (Non tgcs) siblings were 100% PDA defective. n, number of animals scored.