Hua xi kou qiang yi xue za zhi = Huaxi kouqiang yixue zazhi = West China journal of stomatology · 2015
[Influence of 7-dehydrocholesterol reductase gene silencing on the fusion of mouse palatal shelves].
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Wenlin Xiao, Cuizhu Zhuang, Yan Shi, Yaoxiang Xu, Lingfa Xue
Аннотация
OBJECTIVE: RNA interference was applied to knockdown the Dhcr7 gene in mouse embryonic palatal shelves to facilitate understanding of the function of Dhcr7 gene variants in the fusion of palatal shelves.
METHODS: The pAdTrack-CMV-siDhcr7 was constructed using the specific siRNA sequence of Dhcr7 from C57BL/6J mouse. The pAdTrack-CMV- siDhcr7 of positive clones was reconstructed in vitro, and the recombinant adenovirus pAdEasy-1-siDhcr7 of kanamycin resistance was screened. The adenovirus vector DNA was then prepared for transfecting the embryonic palatal shelves. Thirty pairs of embryonic palatal shelves at 13.5 d gestational age were harvested and then randomly divided into the following three groups: normal control group (n = 10), which included palatal shelves inculture medium without cholesterol; blank adenovirus control group (n = 10), which included palatal shelves in culture medium without cholesterol and blank adenovirus; and experimental group (n = 10), which included palatal shelves in culture medium without cholesterol and adenovirus encoding Dhcr7 siRNA. At 48 h after in vitro cultivation, the mRNA and protein of the palatal shelves were obtained for scanning electron microscopy (SEM), reverse transcription polymerase chain reaction (RT-PCR), and Western blot analyses.
RESULTS: SEM showed that the palatal shelves of the normal control and blank adenovirus control groups fused and formed continuous palates, whereas those of the experimental group was almost undeveloped but exhibited large gaps between the two palatal shelves. RT-PCR and Western blot analyses showed that the mRNA and protein of Dhcr7 in the experimental group decreased compared with those in the normal control group with a significant difference (P < 0.05).
CONCLUSION: Results indicate that Dhcr7 gene silencing affects the fusion of palatal shelves. Thus, Dhcr7 gene may serve a function in the normal development of palates.
METHODS: The pAdTrack-CMV-siDhcr7 was constructed using the specific siRNA sequence of Dhcr7 from C57BL/6J mouse. The pAdTrack-CMV- siDhcr7 of positive clones was reconstructed in vitro, and the recombinant adenovirus pAdEasy-1-siDhcr7 of kanamycin resistance was screened. The adenovirus vector DNA was then prepared for transfecting the embryonic palatal shelves. Thirty pairs of embryonic palatal shelves at 13.5 d gestational age were harvested and then randomly divided into the following three groups: normal control group (n = 10), which included palatal shelves inculture medium without cholesterol; blank adenovirus control group (n = 10), which included palatal shelves in culture medium without cholesterol and blank adenovirus; and experimental group (n = 10), which included palatal shelves in culture medium without cholesterol and adenovirus encoding Dhcr7 siRNA. At 48 h after in vitro cultivation, the mRNA and protein of the palatal shelves were obtained for scanning electron microscopy (SEM), reverse transcription polymerase chain reaction (RT-PCR), and Western blot analyses.
RESULTS: SEM showed that the palatal shelves of the normal control and blank adenovirus control groups fused and formed continuous palates, whereas those of the experimental group was almost undeveloped but exhibited large gaps between the two palatal shelves. RT-PCR and Western blot analyses showed that the mRNA and protein of Dhcr7 in the experimental group decreased compared with those in the normal control group with a significant difference (P < 0.05).
CONCLUSION: Results indicate that Dhcr7 gene silencing affects the fusion of palatal shelves. Thus, Dhcr7 gene may serve a function in the normal development of palates.