Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

Pluripotency retention and exogenous mRNA introduction in planarian stem cells in culture

“…The results indicated that live bulk cells exhibited considerably higher NanoLuc signal than that in SiRNeoblasts, even after the depletion of neoblasts by lethal irradiation ( Figures 4 L, 4M, and S6 K), which was consistent with the findings of Hall et al. 33 This result indicated that the NanoLuc signal was derived almost exclusively from somatic cells, and further suggested that planarian neoblasts harbored a means of preventing exogenous nucleic acids before differentiation.…”

“…Culture medium supplements (sodium pyruvate, vitamin, and amino acids, see Table S1 ) were also included in the modified KnockOut DMEM to determine whether they could enhance the mRNA expression. 33 We found that NanoLuc expression levels were higher in SiRNeoblasts cultured in modified KnockOut DMEM with 5% CO 2 modified atmosphere than in cells grown under the same conditions without supplements ( Figure 4 B). To confirm that neoblasts could also be maintained in modified KnockOut DMEM with 5% CO 2 , SiRNeoblasts were stained and their proportion was compared to that of smedwi-1 + cells after one or three days of culture in modified KnockOut DMEM with 5% CO 2 , KnockOut DMEM with 5% CO 2 , and Iso-L15 under ambient conditions.…”

“…A recent study reported that Nanoluciferase ( NanoLuc ) mRNA could be expressed in somatic planarian cells through Viromer or Trans IT transfection. 33 We, therefore, suspected that the high sensitivity and low autofluorescence background of the NanoLuc reporter could provide a tractable approach for visualizing neoblast transgene expression. To confirm that NanoLuc was indeed translated in cultured SiRNeoblasts, we transfected SiRNeoblasts with NanoLuc mRNA using the Trans IT system ( Figure 4 A).…”

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Figure 4 Trans IT can deliver exogenous mRNA into neoblasts and express NLuc in SMEDWI-1 low or SMEDWI-1 - cells (A) Adaption of the RPL15 5′UTR and -3′UTR to NanoLuc luciferase . 33 (B) Expression comparison of NanoLuc luciferase mRNA in 200,000 SiRNeoblasts cultured with or without medium supplements after transfection by Trans IT. Data are represented as mean ± SEM.

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Isolation of planarian viable cells using fluorescence‐activated cell sorting for advancing single‐cell transcriptome analysis

“…In planarians, antibodies against specific cell surface markers used to isolate target cell populations using FACS are limited. However, another planarian species, Schmidtea mediterranea , was able to transfect exogenous genes (Hall et al, 2022). We propose that our sorting method may be useful when combined with gene‐labeling techniques, such as that used for hematopoietic stem cells.…”