Nonviral transfection of mouse calvarial organ in vitro using accell-modified siRNA

Background: Understanding the biology of cranial suture fusion and the precise role of involved molecules implicated in the process will help to identify key factors involved in regulation of suture fusion. Modulation of these key factors may serve as a tissue-engineering technique to replace the traditional surgical procedures for the correction of premature suture fusion. Modulation of gene expression by RNA interference is a widely used technique with high potential. Because there is no available report of calvarial organ transfection in vitro, the authors studied the development of a successful nonviral delivery technique of small inhibitory RNA (siRNA) to an in vitro calvarial organ culture system. Methods: In this study, 19-day-old male CD1 mice were euthanized and parallel craniotomies made through the parietal and frontal calvaria, 2 mm to either side of the sagittal suture, with care taken to preserve the underlying dura mater. Organs grown in vitro in a defined medium were transfected with transforming growth factor-β1-specific Accell-modified siRNA followed by RNA isolation and quantitative polymerase chain reaction analysis. Results: Transfection of a calvarial organ with transforming growth factor-β1-specific Accell-modified siRNA effectively knocks down the mRNA level. Conclusions: Observations from this study indicate that in an in vitro calvarial organ culture system, a specific, efficient, and durable RNA interference activity can be achieved when Accell-modified siRNA is used. In addition to bypassing the need for toxic lipid carriers, the modifications introduced in Accell-modified siRNAs make it more stable and less off-target. This technique can potentially be used for in vivo studies once the initial effect of gene-specific siRNA on in vitro suture fusion has been determined.