FIG. 2.

Both LINE-1 5′ UTR promoters are active in hESCs and hEC cells. (a) L1 expression in pluripotent cells. The cartoon is a schematic of a human LINE-1 element. The relative position and amplification lengths (dashed lines) of the two set of primers (L1-52 and L1-22) (27) used in the quantitative RT-PCR are indicated below the schematic. The position of the amplified region is based on the L1.3 sequence (L19088.1 [71]). Below are shown representative quantitative RT-PCR results for L1 RNA expression using the 5′ UTR (L1-52) or ORF2 (L1-22) primer set analyzed in pluripotent cells and differentiated HeLa cells as a control (35, 36). The graphs show the n-fold changes in L1 expression with respect to H9 hESCs. To normalize the results, we determined the amplification C_T for GAPDH (see Materials and Methods). (b) Pie charts showing the percentages of L1 subfamilies expressed in hEC cells (2102Ep cells) and hESCs (H9 cells). (c) Cartoon of an RC-L1 (top) with two clones generated that contain the 5′ UTR of L1.3 in the antisense (left, 5AS-FF) or the sense (right, 5S-FF) orientation. The white boxes indicate the ORF coding for firefly luciferase, and black lollipops represent polyadenylation signals. (d) Quantification of the activities of the sense and antisense L1Hs promoters in hESCs and 2102Ep hEC cells. The graph shows the firefly luciferase activity in LUs, normalized to Renilla luciferase activity, which served as an internal control for transfection efficiency (see Materials and Methods), of the sense (5S) and antisense (5AS) promoters in hEC cells (2102Ep, dark bar graph), hESCs (H13B (gray bar graph), and H7 cells (white bar graph) measured in triplicate (the standard deviation of the assay is indicated in each graph). LUs from these assays are shown in Fig. 2 posted at http://www.juntadeandalucia.es/fundacionprogresoysalud/repositorio/files/0000/0080/16._MCB00561_SupInfo_final.pdf. UTF, untransfected.