key: cord-0332766-xv4j3u49
authors: Hall, Richard Nelson; Weill, Uri; Khariton, Margarita; Leal-Ortiz, Sergio; Drees, Leonard; Chai, Chew; Xue, Yuan; Rosental, Benyamin; Quake, Stephen R.; Sánchez Alvarado, Alejandro; Melosh, Nicholas A.; Fire, Andrew; Rink, Jochen C.; Wang, Bo
title: Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection
date: 2021-04-20
journal: bioRxiv
DOI: 10.1101/2021.04.20.440701
sha: 367c515a5b57e5a566e626d3101d4c25bf7bf504
doc_id: 332766
cord_uid: xv4j3u49

Planarians have long been studied for their regenerative abilities, but they possess limited genetic tools due to challenges in gene delivery, expression, and detection, despite decades of work. We developed a toolbox for heterologous protein expression in planarian cells and in live animals. Specifically, we identified and optimized nanotechnological and chemical transfection methods to efficiently deliver mRNA encoding nanoluciferase into somatic cells, including planarian adult stem cells (neoblasts). The use of a luminescent reporter allowed us to quantitatively measure protein expression through spectroscopy and microscopy, thus overcoming the strong autofluorescent background of planarian tissues. Using this platform, we investigated the use of endogenous untranslated region (UTR) sequences and codon usage bias to post-transcriptionally alter gene expression. Our work provides a strong foundation for advancing exogenous gene expression and for the rapid prototyping of genetic constructs to accelerate the development of transgenic techniques in planarians.

In order to establish mRNA expression in the asexual strain of S. mediterranea, we sought to 114 identify an efficient and species-agnostic platform for delivering genetic material into primary 115 planarian cells. We selected nanostraws, which combine the robustness of mechanical methods 116 like microinjection with the throughput of electrical methods like electroporation (Tay & 117 Melosh, 2019). Figure 1C) . Therefore, we performed flow cytometry analysis to quantify fluorescence signal 143 and found, even in this relatively uniform cell population, a broad distribution of fluorescence 144 intensity, spanning three orders of magnitude in both experimental and negative conditions 145 ( Figure 1D ). This highly variable autofluorescence in our measurements highlights the 146 difficulties of using fluorescent reporters. Since some cells genuinely exhibit brighter 147 fluorescence than others, false positives may be common and true positives could be obscured by 148 the broad autofluorescent background such that H2B-mScarlet expression cannot be 149 unambiguously assessed. Together, these results compelled us to seek an alternative non-150 fluorescent reporter to quantify gene expression in planarian cells. 169 With a validated reporter construct in hand, we next sought to identify a more broadly accessible Table 2 ). While most transfections had 180 little to no effect, Viromer and Trans-IT (Mirus) reagents consistently achieved luminescent 181 signals 100 to 1,000-fold above the negative control (Figure 2A) . Table 1 ). We also 252 included a Nluc sequence optimized for mammalian expression as a reference for comparison.

Genes with higher CAI values tended to have lower GC content, reflecting the A/T bias in S. We next asked whether Nluc expression in live worms was sufficient for luminescence imaging. Concentrate kit (Zymo). The purified fragments were ligated together using T4 DNA Ligase 509 (NEB) to create pNHT7 (Figure 3 -figure supplement 1A) . 1 µL BsaI-HF to produce pNHT7::GOI (Figure 3 -figure supplement 1B) .

Finally, to insert a reporter between the 5' and 3' UTRs, we amplified pNHT7::GOI with 519 outward facing primers (BW-NH-128-139) which bind to the end and beginning of the 5' and 3' 520 UTRs respectively containing the BsaI restriction sites. The reporter was then amplified to 521 append compatible BsaI restriction sites using primers BW-NH-174-185 and inserted between 522 the two UTR sequences via a golden gate reaction. The resulting plasmid was amplified with 523 M13 forward and M13 reverse primers to produce linear template for in vitro transcription 524 reactions (Figure 3 -figure supplement 1C) . which dips into the cell culture medium from above, the anode, which sits just below the buffer 832 and mRNA to be delivered, and the nanostraw membrane, which is inserted atop the buffer and show that ~94% were positive for the neoblast marker piwi-1. Of these piwi-1 + cells, they were 849 38 split between neoblast subpopulations including epidermal, muscle, and gut progenitors 850 (N_epidermal, N_muscle, and N_gut), and clonogenic neoblasts (cNeoblasts, tgs + ). 

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