Fig. 9. Evaluation of RNAi vectors in 293T cells with GFP-fusion proteins. 293T cells (8 ´ 10⁵ cells in 60-mm dish) were cotransfected with three DNA constructs, a GFP-fusion protein expression vector (1 μg), an RNAi vector (3 μg), and pCAG-HcRed (1 μg) as a control and analyzed 72 h after transfection. The GFP-fusion proteins, Crx:GFP (a-d), Nrl:GFP (i-l), and GAPDH:GFP (q-t) were expressed under the control of the CAG promoter. Note that Crx:GFP and Nrl:GFP fusion proteins were localized in the cell nucleus, whereas GAPDH:GFP fusion protein was localized in the cytosol. The RNAi vector was a pBS/U6 empty vector (U6; a, e, i, m, q, and u) or pBS/U6 carrying a DNA template that directs synthesis of mCrx siRNA (b, f, j, n, r, and v), mNrl siRNA (c, g, k, o, s, and w) or mGAPDH siRNAs (d, h, l, p, t, and x). For construction of cDNAs encoding Crx:GFP, Nrl:GFP, and GAPDH:GFP fusion proteins, AgeI sites were created by PCR at the end of coding regions of mCrx, mNrl and mGAPDH, respectively, and ligated with the GFP cDNA excised from pEGFP-N1 (Clontech) with AgeI and NotI. These cDNAs were cloned into pCAGGS with modified multiple cloning sites. For construction of pCAG-HcRed, a coding region of HcRed was excised from pHcRed1-N1 (Clontech) and cloned into pCAGGS with modified multiple cloning sites.