The shRNA against SOD1G93A
(G93Ahp) contains a stem that is homologous to SOD1G93A
mRNA but has a mismatched nucleotide with SOD1WT
at the middle of the stem (Fig. A). When transfected into cultured cells, this shRNA selectively inhibited the expression of SOD1G93A
but did not affect the expression of SOD1WT
) (also see below). However, the RNAi efficacy of this shRNA was relatively poor (see below). We therefore sought to increase the potency of this shRNA by increasing its expression. We modified the U6 promoter by placing the enhancer from the CMV immediate-early promoter near the U6 promoter, either upstream or downstream from U6G93Ahp and in either forward or reverse orientation (Fig. B).
Figure 1 Design of hairpin constructs against mutant SOD1G93A. (A) Nucleic acid sequence surrounding the mutation site of SOD1G93A. Notice the G (bold) in the antisense strand of the hairpin that matches with the C (bold) in SOD1G93A but mismatches with a G in (more ...)
We co-transfected each of the seven constructs containing various combinations of U6 promoter, G93Ahp and CMV enhancer, with a target construct that encoded a SOD1G93A
and GFP fusion protein (SOD1G93A
GFP), into human 293 cells. Northern blot analysis demonstrated that addition of the CMV enhancer near U6G93Ahp in all four configurations (Fig. ) increased the expression of G93Ahp (Fig. ). Deletion of the distal sequence element (DSE), an obligatory component of the U6 promoter (25
), abolished expression of G93Ahp even in the presence of the enhancer (Fig. ). Quantification of SOD1G93A
GFP expression by GFP fluorescence using a fluorometer (31
) showed that, compared with SOD1G93A
GFP alone transfection (Fig. A and B, 1), G93Ahp produced by the unmodified U6 promoter (U6G93Ahp) inhibited SOD1G93A
GFP expression modestly (Fig. A and B, 2). Attaching the CMV enhancer in all four configurations to U6G93Ahp (Fig. B) enhanced the inhibition of SOD1G93A
GFP expression to a similar degree (Fig. A and B, 3–6). Deletion of the DSE abolished the inhibition of SOD1G93A
GFP expression (Fig. A and B, 7). Finally, U6 promoter directed synthesis of mismatched shRNA did not show any inhibitory activity towards the target gene (Fig. A and B, 8).
Figure 2 Northern blot detecting the G93Ahp transcripts. Total RNA was extracted from human 293 cells doubly transfected with SOD1G93AGFP and various U6G93Ahp constructs (Fig. B). G93Ahp was detected using a 32P-labeled 21 nt RNA probe complementary (more ...)
Figure 3 CMV enhancer increases the inhibition of target gene expression. (A) Fluorometer measurement of GFP fluorescence in lysates from the 293 cells transfected with SOD1G93AGFP and various U6G93Ahp constructs (Fig. B). (B) Average peak GFP (more ...)
Western blot using a polyclonal anti-SOD1 antibody confirmed the above finding and, furthermore, showed that the enhanced synthesis of G93Ahp only inhibited SOD1G93AGFP expression but did not affect the endogenous human SOD1 levels (Fig. C), indicating that the high levels of G93Ahp expression do not affect the specificity of G93Ahp for the mutant SOD1G93A.
Our results demonstrate that the CMV enhancer can enhance U6 promoter activity and increase the production of shRNA. This modified promoter may be useful where limited choices of shRNA sequences preclude the selection of a highly efficient RNAi target region and, therefore, could be used for selective inhibition of mutant gene expression in vitro and in vivo and to develop therapies for diseases caused by dominant, gain-of-function gene mutations.