Abstract
BackgroundAchieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. To better understand the molecular mechanisms involved in skull growth, and in abnormal growth conditions, such as craniosynostosis, caused by premature bony fusion, it is essential to be able to easily introduce genes into primary bone forming cells to study their function.ResultsA comparison of several lipid-based techniques with two electroporation-based techniques demonstrated that the electroporation method known as nucleofection produced the best transfection efficiency. The parameters of nucleofection, including cell number, amount of DNA and nucleofection program, were optimized for transfection efficiency and cell survival. Two different genes and two promoter reporter vectors were used to validate the nucleofection method and the responses of human primary suture mesenchymal cells by fluorescence microscopy, RT-PCR and the dual luciferase assay. Quantification of bone morphogenetic protein (BMP) signalling using luciferase reporters demonstrated robust responses of the cells to both osteogenic BMP2 and to the anti-osteogenic BMP3.ConclusionsA nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for in vitro studies of gene and protein function in human skull growth. Human primary suture mesenchymal cells exhibit robust responses to BMP2 and BMP3, and thus nucleofection can be a valuable method for studying the potential competing action of these two bone growth factors in a model system of cranial bone growth.
Highlights
Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research
Lipid-based methods commonly used to transfect transformed cells are generally ineffective with primary cells and achieving efficient transfection of primary cells is a common problem in biomedical research
None of the transfection agents, except endofectine and metafectine, showed any significant cell death as observed by light microscopy but the percentage of Green Fluorescent Protein (GFP)-positive cells determined by flow cytometry indicated that these agents produced only 1-4 % transfection efficiency (Table 1)
Summary
Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. They are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. The ability to transfect DNA into mammalian cells is vital in biomedical research, in studies of mechanistic understanding and clinical application. Non-viral electroporation of genes into primary cells can provide a simple and efficient alternative but, typically, primary cells of different origins cannot necessarily be transfected using the same conditions, which need to be optimized [5,6,7,8,9,10]
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