Sample Abstract - Note: “beta” will read as ß in the abstract book.

MODULATION OF TGF-beta1 GENE EXPRESSION IN VIVO DURING MOUSE CALVARIAL SUTURE FUSION

Yu P, Gosain AK, Khanna AK

Medical College of Wisconsin, Milwaukee, WI

Introduction: TGF-beta1 is actively expressed during mouse calvarial suture fusion. We have recently demonstrated that TGF-beta antibody inhibited fusion of the posterior frontal (PF) suture in vitro. The present study was performed to investigate whether modulation of PF suture fusion can be achieved in vivo, by blocking TGF-beta1 gene expression using antisense containing plasmid DNA.
Methods: TGF-beta1 cDNA was cloned and inserted in opposite direction into the expression cassette of the plasmid vector RLDN. This TGF-beta1 antisense containing 10 mg plasmid DNA in 50 ml
lipofectamine was then injected into the subgaleal layer along the frontal suture of 22 day old mice under anesthesia (n=3). For control groups, 50 ml lipofectamine alone was used (n=3). The PF sutures were harvested at various time points and examined by histology with histometric analysis,
immunohistochemistry, and reverse transcription and PCR for the detection of mRNA.
Results: All animals survived after injection of antisense or lipofectamine. Histology showed that PF suture fusion was significantly delayed in the antisense group compared to control. By postnatal day 45, suture fusion was complete in control animals. There was a 70% inhibition of suture fusion (new bone area) in antisense groups measured with an imaging system. Four days after antisense injection (age 26 days), mRNA expression for TGF-beta1 was 77% lower in the antisense group (2.3±1.8) than in the control group (9.8±3.1). Immunohistochemistry studies also showed decreased TGF-beta1 expression in the antisense group.
Conclusion: We have successfully established an in vivo animal model of blocking TGF-beta1 gene expression in mouse calvarial sutures using antisense containing plasmid DNA. Such a DNA transfection appears to inhibit fusion of the PF suture, further establishing the role of TGF-beta1 in calvarial suture fusion. These data indicate that modulating TGF-beta1 gene expression in vivo can alter the natural history of cranial suture fusion.

 

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