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Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/74313

Type: Journal article
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
Author: Dwivedi, Prem Prakash
Anderson, Peter John
Powell, Barry Crampton
Citation: BMC Biotechnology, 2012; 12:45
Publisher: BioMed Central Ltd.
Issue Date: 2012
ISSN: 1472-6750
School/Discipline: School of Paediatrics and Reproductive Health : Paediatrics
Statement of
Responsibility: 
Prem P Dwivedi, Peter J Anderson and Barry C Powell
Abstract: Background: 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 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. Results: 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 antiosteogenic BMP3. Conclusions: A 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.
Keywords: Transfection; Nucleofection; Skull; Bone; Primary cell culture; Mesenchymal; BMP2; luciferase
Description: Extent: 9p.
Rights: © 2012 Dwivedi et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
RMID: 0020121910
DOI: 10.1186/1472-6750-12-45
Appears in Collections:Paediatrics Publications
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