IADR Abstract Archives

Knockdown of Gene Expression in Telomerase Immortalized Gingival Keratinocytes

Objectives: In health, oral commensal bacteria maintain a homeostasis with the oral epithelium. Disruption of homeostasis provides pathogenic species an opportunity to become more prevalent and initiate an inflammatory response. Many functions of host-commensal interactions in controlling pathogenic oral bacteria have yet to be examined. We find decreased bacterial adherence of commensals and adherence/invasion of pathogens to telomerase immortalized gingival keratinocytes (TIGKs) after an initial challenge with the commensal, Streptococcus mitis. Coincidently, TIGKs display upregulation of membrane-associated mucins (MAMs). Towards interrogating surface proteins/glycoproteins in host-commensal interactions and innate immunity, we set out to optimize conditions to knockdown TIGK transcripts of the MAM, MUC1.
Methods: Conditions were optimized for maximum concentration of transfection reagent (Dharmafect1) that maintains cell viability. Increasing amounts of Dharmafect1 (0, 0.25, 0.5, 0.75, 1.0, 1.25µl) were added to culture medium (500µl) of TIGKs grown to 50% confluency. After 0, 48 and 96h of incubation, cells were evaluated for confluency under phase-contrast microscopy. Also, three fields (200x) were assayed for percentages of apoptotic (Annexin V staining) and necrotic cells (propidium iodide staining). The three highest concentrations of Dharmafect1 with minimal effects on cell viability were then tested with TIGKs at 80% confluency for knockdown of MUC1 transcripts after 48h and 96h (TaqMan Q-PCR assay) using 25nM siRNA (Thermo Scientific SMARTpool ON-TARGETplus MUC1 siRNAs) and Non-targeting Pool siRNAs (control).
Results: At 96h, confluency decreased with 0.75µl Dharmafect1 and above. Confluency increased under all conditions at 48h, but the increase was attenuated with increasing Dharmafect1 and mostly plateaued at 1µl and 1.25µl. Necrosis and apoptosis were below control levels with 1µl or less of Dharmafect1. Knockdown of MUC1 transcripts ranged from 60% to 81% (maximal at 48h with 1µl Dharmafect1).
Conclusions: Collective results suggest 1µl Dharmafect1 with 25nM siRNA is a good starting point in optimizing TIGK gene knockdown.
Division: IADR/AADR/CADR General Session
Meeting: 2015 IADR/AADR/CADR General Session (Boston, Massachusetts)
Location: Boston, Massachusetts
Year: 2015
Final Presentation ID: 3147
Abstract Category|Abstract Category(s): Periodontal Research - Pathogenesis
Authors
  • Faby, Eric  ( University of Florida College of Dentistry , Gainesville , Florida , United States )
  • Cuadra-saenz, Giancarlo  ( University of Florida College of Dentistry , Gainesville , Florida , United States )
  • Culp, David  ( University of Florida College of Dentistry , Gainesville , Florida , United States )
  • Support Funding Agency/Grant Number: NIDCR, T90 DE021990
    Financial Interest Disclosure: NONE
    SESSION INFORMATION
    Poster Session
    Genetics, Gene Expression and Signaling
    Friday, 03/13/2015 , 03:30PM - 04:45PM