OBJECTIVETo explore the silencing effect of short hairpin RNA (shRNA) on the CD28 of mice T lymphocytes by CD28-shRNA expressing plasmid evaluate the interfering effects (chronology and stability) mediated by shRNA and select out the most efficient CD28 shRNA sequence.

METHODSThree CD28 specific and one non-specific shRNA expressing plasmids were constructed and then transfected separately into mice spleen T lymphocytes. Non-transfected cells and non-specific shRNA were taken as controls. Inhibitory effect of CD28 shRNA was demonstrated by real-time quantitative PCR and Western blots. The sequence of the highest RNA interference (RNAi) efficacy was screened.

RESULTS(1) CD28 shRNA expressing plasmids were successfully constructed; (2) Three CD28 specific shRNAs effectively inhibited the expression of CD28 at the mRNA and protein levels, and there was a statistically significant difference comparing with the controls (P < 0.01): The copies of CD28 in mice spleen cells at the mRNA levels were persistently decreased by 99.62%, 99.89% and 99.80% respectively after 20 days, and so did at the protein level [(84.90 +/- 0.65)%, (96.49 +/- 0.03)%, (91.76 +/- 0.32)% respectively]. The highest inhibitory rate was in CD28 shRNA-2 group.

CONCLUSIONS(1) Specific shRNA can mediate long-term and stable silencing effects on CD28 gene; (2) shRNAs matching different sites of CD28 gene exert differential inhibitory effects.