Figure 5

In vivo effects of direct transfer of p21 gene in mice. A: modulation of p21 gene expression: Group of mice (n = 24) were injected intramuscularly with either empty vector, CD3p21 sense, CD3p21 Antisense plasmid DNA (n = 8 each). Modulation of p21 was monitored by p21 mRNA expression using Real Time PCR analysis of lymphocytes. Results from representative 2 animals from each group are shown. The mRNA expresion of both p21 and CD3 is seen only in mice injected with CD3p21 sense plasmid DNA, whereas as expected only CD3 mRNA expression is detectable in mice injected with empty vector plasmid DNA. These results demonstrate the ability of CD3p21 sense plasmid DNA to modulate p21 gene in T lymphocytes. B: In vivo effect of p21 overexpression on lymphocyte proliferation. Lymphocytes from mice injected with empty vector plasmid DNA (control mice n = 7), CD3p21 sense and antisense plasmid DNA (n = 7) were isolated and the proliferation in response to murine anti-CD3 monoclonal antibody was quantified using ³H-thymidine uptake assay. The lymphocytes from mice injected with CD3p21 sense plasmid DNA proliferated significantly (p < 0.01) less and with CD3p21 antisense plasmid DNA proliferated significantly (p < 0.001) more compared to the control mice. These results demonstrate that the direct gene modulation in T cells can influence their proliferation and inflammation. C: In vivo effect of p21 modulation on TNF-α and IFN-γ mRNA expression: RNA from lymphocytes obtained from mice injected with CD3Sp21 and CD3ASp21 plasmid DNA were isolated, reverse transcribed and analyzed for TNF-α and IFN-γ mRNA expression using Real Time PCR assay. Fold increase of mRNA expression in lymphocytes from CD3Antisensep21 injected mice was calculated with respect to the CD3Sensep21 DNA injected mice.