Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3
<p>Abstract</p> <p>Background</p> <p>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 bo...
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| Format: | Article |
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BMC
2012-08-01
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| Series: | BMC Biotechnology |
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| Online Access: | http://www.biomedcentral.com/1472-6750/12/45 |
| _version_ | 1818542430099603456 |
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| author | Dwivedi Prem P Anderson Peter J Powell Barry C |
| author_facet | Dwivedi Prem P Anderson Peter J Powell Barry C |
| author_sort | Dwivedi Prem P |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs <it>in utero</it> 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.</p> <p>Results</p> <p>A 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.</p> <p>Conclusions</p> <p>A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for <it>in vitro</it> 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.</p> |
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| language | English |
| last_indexed | 2024-12-11T22:21:57Z |
| publishDate | 2012-08-01 |
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| spelling | doaj.art-f577ece61b2c4a819f0172e39150bc242022-12-22T00:48:24ZengBMCBMC Biotechnology1472-67502012-08-011214510.1186/1472-6750-12-45Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3Dwivedi Prem PAnderson Peter JPowell Barry C<p>Abstract</p> <p>Background</p> <p>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. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs <it>in utero</it> 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.</p> <p>Results</p> <p>A 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.</p> <p>Conclusions</p> <p>A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for <it>in vitro</it> 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.</p>http://www.biomedcentral.com/1472-6750/12/45TransfectionNucleofectionSkullBonePrimary cell cultureMesenchymalBMP2luciferase |
| spellingShingle | Dwivedi Prem P Anderson Peter J Powell Barry C Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3 BMC Biotechnology Transfection Nucleofection Skull Bone Primary cell culture Mesenchymal BMP2 luciferase |
| title | Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3 |
| title_full | Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3 |
| title_fullStr | Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3 |
| title_full_unstemmed | Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3 |
| title_short | Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3 |
| title_sort | development of an efficient non viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to bmp2 and bmp3 |
| topic | Transfection Nucleofection Skull Bone Primary cell culture Mesenchymal BMP2 luciferase |
| url | http://www.biomedcentral.com/1472-6750/12/45 |
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