Barela Hudgell MA and Smith LC (2022), Lipofection mediated transfection fails for sea...

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PLoS One 2022 Jan 01;175:e0267911. doi: 10.1371/journal.pone.0267911.

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Lipofection mediated transfection fails for sea urchin coelomocytes.

Barela Hudgell MA , Smith LC .

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Molecular cloning, gene manipulation, gene expression, protein function, and gene regulation all depend on the introduction of nucleic acids into target cells. Multiple methods have been developed to facilitate such delivery including instrument based microinjection and electroporation, biological methods such as transduction, and chemical methods such as calcium phosphate precipitation, cationic polymers, and lipid based transfection, also known as lipofection. Here we report attempts to lipofect sea urchin coelomocytes using DOTAP lipofection reagent packaged with a range of molecules including fluorochromes, in addition to expression constructs, amplicons, and RNA encoding GFP. DOTAP has low cytotoxicity for coelomocytes, however, lipofection of a variety of molecules fails to produce any signature of success based on results from fluorescence microscopy and flow cytometry. While these results are negative, it is important to report failed attempts so that others conducting similar research do not repeat these approaches. Failure may be the outcome of elevated ionic strength of the coelomocyte culture medium, uptake and degradation of lipoplexes in the endosomal-lysosomal system, failure of the nucleic acids to escape the endosomal vesicles and enter the cytoplasm, and difficulties in lipofecting primary cultures of phagocytic cells. We encourage others to build on this report by using our information to optimize lipofection with a range of other approaches to work towards establishing a successful method of transfecting adult cells from marine invertebrates.

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	Fig 1. The percentage of viable coelomocytes over 36 hours does not change when incubated with DOTAP.Cell viability was gauged using propidium iodide exclusion after incubation with DOTAP. Coelomocyte suspensions (1.5 X 105) were treated with 0 to 30 μL of DOTAP and tested for viability at 1, 2, 3 and 36 hours (hr). The salinity of the medium was adjusted for some samples by adding NaCl to compensate for the addition of DOTAP in HBS.
	Fig 2. Green fluorescence in coelomocytes is increased in samples lipofected with expression constructs to drive GFP expression.Flow cytometry histograms for green fluorescence are shown for replicate samples of coelomocytes incubated with DOTAP lipoplexes with one of the GFP expression constructs (A-I; green), or with DOTAP liposomes without DNA (J-L; red), or were not treated (blue). Histograms of lipofected coelomocytes are overlaid on histograms of untreated control cells.
	Fig 3. Coelomocytes lipofected with the pONY_HE_GFP-X expression construct or the amplicon show low levels of GFP expression.(A-F) A few selected cells show green fluorescence after lipofection with the pONY_HE_GFP-X expression construct. (G-I) Similarly, only a few cells show green fluorescence after lipofection with the HE_GFP-X amplicon. The white arrows in (A), (D), and (G) indicate GFP expressing cells in (B), (E), and (H). Scale bars in the merge images apply to the other panels in the same row.
	Fig 4. Coelomocytes lipofected with mRNA encoding GFP or mCherry result in very few examples of fluorescent cells.(A-C) Coelomocytes lipofected with GFP sense strand mRNA. (D-F) Coelomocytes lipofected with mCherry sense strand mRNA. (B) Selected coelomocytes show GFP green fluorescence. (E) Selected coelomocytes show mCherry fluorescence. The scale bar in (B) applies to panels (A) and (C). The scale bar in (E) applies to panels (D) and (F). Results for all fluorescent cell instances and controls are shown in Table 4.

References [+] :

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