Department of Biological Sciences, George Washington University, Washington, DC, United States of America.
PLoS One. 2022 May 6;17(5):e0267911. doi: 10.1371/journal.pone.0267911. eCollection 2022.
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.
分子克隆、基因操作、基因表达、蛋白质功能和基因调控都依赖于将核酸导入靶细胞。已经开发了多种方法来促进这种递呈，包括基于仪器的显微注射和电穿孔、转导等生物学方法以及磷酸钙沉淀、阳离子聚合物和脂质转染等化学方法，也称为脂质体转染。在这里，我们报告了使用 DOTAP 脂质体试剂转染海胆体腔细胞的尝试，该试剂包装了一系列分子，包括荧光染料，以及表达构建体、扩增子和编码 GFP 的 RNA。DOTAP 对体腔细胞的细胞毒性较低，然而，各种分子的脂质体转染都没有根据荧光显微镜和流式细胞术的结果产生任何成功的迹象。虽然这些结果是负面的，但重要的是要报告失败的尝试，以便其他进行类似研究的人不会重复这些方法。失败可能是由于体腔细胞培养液的离子强度升高、内体溶酶体系统中脂质体复合物的摄取和降解、核酸无法从内体小泡逃逸并进入细胞质以及吞噬细胞原代培养物的脂质体转染困难所致。我们鼓励其他人在本报告的基础上，利用我们的信息，通过一系列其他方法优化脂质体转染，以努力建立一种成功的转染海洋无脊椎动物成体细胞的方法。
