Lipofection mediated transfection fails for sea urchin coelomocytes

Abstract

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.

Transfection of Sponge Cells and Intracellular Localization of Cancer-Related MYC, RRAS2, and DRG1 Proteins

“…It is one of the most important procedures for molecular cloning, gene manipulation, and gene expression and regulation, as well as protein function and regulation studies. Transfection can be performed biologically (using a viral vector, and then it is called transduction), physically/mechanically (e.g., gene gun, particle bombardment, microinjection, and laser-based transfection), chemically (e.g., calcium phosphate precipitation, cationic polymers, and lipid-based transfection, known as lipofection), or combined (e.g., electroporation) [ 1 , 2 ]. Although there is constant development and progress in transfection techniques, each method has its own advantages and disadvantages, depending on the experimental goal, cell, or tissue type.…”

“…For example, the presence or absence of serum in the cell culture medium and the ratio of liposome-to-DNA to avoid toxicity are important factors that need to be tested and evaluated before the actual experiment [ 6 ]. Electroporation induces the formation of nanometer-size reparative pores in cell membranes by exposing cells to a brief high-voltage electric field and introducing nucleic acids into cells through these pores [ 1 ]. Although this method might alter the cell phenotype, it is useful for transfecting a large number of cells at once and often results in higher transfection efficiency and lower cell death, especially within cells that are generally resistant to other transfection methods [ 4 , 8 ].…”

Exploration of mRNA nanoparticles based on DOTAP through optimization of the helper lipids

“…[9] DOTAP at a concentration of ⦤150 g L −1 is non-cytotoxic to mammalian cells (Roche Diagnostics). [10] Recently, LNPs based on DOTAP have been used in preclinical and clinical settings widely. [3,[11][12][13][14][15] For example, an efficient lung-specific mRNA delivery platform known as five-element nanoparticles (FNPs) has been developed by combining PBAEs and DOTAP.…”