Hoxb13 Gene Silencing by Small Interference RNA

Deans

Robert Uchin – College of Dental Medicine Andrés Malavé – College of Pharmacy

Award Date

1-1-2006

Abstract

Severe scarring is one of the most serious complications after cleft lip and palate surgeries. Homeobox gene Hoxb13 has been reported to play a role in scarless wound healing process. Recent adult wound healing studies using an adult Hoxb13 knockout mouse model indicated that cutaneous wound healing is fetal-like with reduced scar formation. Our long-term goal is to improve wound healing and reduce scar formation after cleft lip surgery. The objective of this proposed project is, primarily, to develop a Hoxb13 gene silencing tool. It is our central hypothesis that an optimized small interference RNA (siRNA), combined with proper delivery tools will be able to reduce Hoxb13 gene expression, thus reducing scar formation. SiRNA oligos are usually 19-22 base pairs in length and when introduced into a cell, inhibit specific gene expression in a sequence-dependent fashion. It is thus far the most effective and feasible method in inhibiting specific gene expression. In this study, we will obtain an optimal siRNA oligo against Hoxb13. The Hoxb13 gene sequence will be inputted into "SiRNA Target Finder" (www.ambion.com) which will aid in designing siRNA oligos. A Hoxb13-GFP (Green Fluorescent Protein) fusion gene will be used as the target to be suppressed by the siRNA oligos. Suppression of Hoxb13 gene expression will lead to the suppression of GFP expression since it expresses as a fusion protein. Suppression activity will be determined by the reduction in green fluorescence and confirmed by Northern blot at the mRNA level. In addition, we will use the biodegradable nanoparticles to deliver siRNA. The delivery efficiency will be confirmed by fluorescence intensity since the siRNA will be labeled with a fluorescencent molecule. Silencing activity will be determined as described above. In the future, we may use nanoparticles to deliver the Hoxb13 siRNA into a dermal wounding area in situ.

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