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. 2012 Jul;37(7):1578-83.
doi: 10.1007/s11064-012-0753-x. Epub 2012 Mar 25.

Efficient down-regulation of glia maturation factor expression in mouse brain and spinal cord

Smita Zaheer  1 , Yanghong Wu, Xi Yang, Ramasamy Thangavel, Shailendra K Sahu, Asgar Zaheer
Affiliations

Efficient down-regulation of glia maturation factor expression in mouse brain and spinal cord

Smita Zaheer et al. Neurochem Res. 2012 Jul.

Abstract

Long-lasting siRNA-based down-regulation of gene of interest can be achieved by lentiviral-based expression vectors driving the production of short hairpin RNA (shRNA). We investigated an attractive therapeutic approach to target the expression of proinflammatory GMF by using lentiviral vector encoding GMF-specific shRNA to reduce GMF levels in the spinal cord and brain of mice. To determine the effect of GMF-shRNA on GMF protein levels, we performed quantitative ELISA analysis in brain and in thoracic, cervical and lumbar regions of spinal cord from mice followed by GMF-shRNA (G-shRNA) or control shRNA (C-shRNA) treatments. Our results show a marked reduction of GMF protein levels in brain and spinal cord of mice treated with GMF-shRNA compared to control shRNA treatment. Consistent with the GMF protein analysis, the immunohistochemical examination of the spinal cord sections of EAE mice treated with GMF-shRNA showed significantly reduced GMF-immunoreactivity. Thus, the down-regulation of GMF by GMF-shRNA was efficient and wide spread in CNS as evident by the significantly reduced levels of GMF protein in the brain and spinal cord of mice.

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Figures

Figure 1
Figure 1
Expression of β-galactosidase. The ventral, dorsal and coronal views of brain of mouse 48 h after injection with AdCMV-βGal. Dark blue staining showing β-galactosidase expression on the major cerebral arteries. Transgene expression was also observed on cerebellum and spinal cord.
Figure 2
Figure 2
Light microscopy of β-galactosidase histochemistry of mouse brain after 48 h after injection of the AdCMV-βGal shows gene expression in cortex (A, E), amygdala (B, F), cerebellum (C, G), and spinal cord (D, H). (Magnification A–D, 10×; E–H, 20×)
Figure 3
Figure 3
Hippocampus section showing the β-gal histochemistry (counterstained with neutral red, magnification A, 20×; B, 40×).
Figure 4
Figure 4
LV-shRNA-mediated down-regulation of GMF in brain and spinal cord. GMF protein levels were measured by quantitative ELISA on day 24 p.i. *, p < 0.001 for GMF-shRNA versus C-shRNA
Figure 5
Figure 5
(5A) Immunostaining of GMF in spinal cord sections of mice injected with GMF-shRNA. (A) MOG-immunized (day 16), (B) MOG +.G-shRNA (day 16), and (C) MOG + G-shRNA (day 24). (5B) Number of GMF-immunopositive cells in MOG-immunized, MOG + GMFshRNA (day 16), and MOG + GMFshRNA (day 24) groups.
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