An EGFP retrotransposition cassette to detect near real time L1 retrotransposition
We created a retrotransposition cassette consisting of the EGFP gene, the CMV immediate early promoter, the TK poly(A) signal and a γ-globin intron in the opposite orientation to the EGFP gene. The EGFP retrotransposition cassette was then cloned into the 3′-UTR of various L1 elements. Before retrotransposition, EGFP transcripts from the CMV promoter contain the γ-globin intron in the opposite orientation and cannot produce functional EGFP protein. EGFP expression and concomitant fluorescence under UV light can only occur when the γ-globin intron is removed by splicing during a retrotransposition event (Fig. A).
Figure 1 (A) The L1 element consists of the 5′- and 3′-UTRs and two ORFs. The EGFP retrotransposition cassette is cloned into the L1 3′-UTR in the antisense orientation. The cassette consists of the CMV immediate early promoter (pCMV), (more ...)
We cloned L1 elements containing the EGFP retrotransposition marker into the pCEP4 episomally replicating mammalian expression vector, which contains a hygromycin resistance gene, or into a pCEP-based vector in which the hygromycin resistance gene was replaced by the puromycin resistance gene. These constructs were then transfected into HeLa cells. Cells transfected with the L1 constructs were selected by applying the appropriate antibiotic (hygromycin or puromycin) 24 h post-transfection. After eliminating untransfected cells, we determined relative retrotransposition frequencies by FACS (Fig. B). We were able to detect fluorescent cells, presumably containing retrotransposition events, by microscopy as early as 48 h post-transfection (Fig. C).
We assessed whether the γ-globin intron was appropriately removed by splicing. First, we picked several independent EGFP-expressing colonies from the retrotransposition assay, expanded the colonies and isolated genomic DNA. Then we performed PCR using intron-flanking primers, which indicated that in every case the intron had been removed as expected (Fig. ). We confirmed precise splicing of the γ-globin intron by sequencing several of the PCR products (data not shown).
Figure 2 Genomic DNA from independent EGFP-expressing clones was isolated and used as template in PCR to determine if the γ-globin intron had been removed by splicing. The PCR products were separated on a 0.7% agarose gel containing ethidium bromide. (more ...)
Retrotransposition is not detected in cell culture during the first 48 h post-transfection, then proceeds at a continuous high rate for at least 16 days
We performed two types of experiments to determine retrotransposition kinetics in cell culture. In the first type, we used constructs encoding EGFP-tagged L1 elements and the hygromycin resistance gene. As observed by microscopy, untransfected cells were almost completely eliminated after 7 days of hygromycin selection. More than 99% of the remaining untransfected cells were excluded by live–dead gating, confirming elimination of untransfected cells by 8 days post-transfection. None of the untransfected control cells analyzed was fluorescent above the threshold. To sample time points earlier than 8 days post-transfection, we performed a second type of experiment using constructs encoding the EGFP-tagged L1 elements and the puromycin resistance gene. Untransfected cells were nearly completely eliminated by puromycin selection 3 days post-transfection and >98% of the remaining untransfected cells were excluded by live–dead gating. As before, none of the untransfected cells analyzed were fluorescent above the threshold. Using fluorescence microscopy, retrotransposition was not detected until 48 h post-transfection in any of the experiments. Fluorescent cells were detected between 48 and 72 h post-transfection among the cells transfected with any construct that contained the tagged L1RP or L1.3 elements.
Following the 48 h period of no detectable retrotransposition, a continuous high rate of retrotransposition was observed. In experiments using hygromycin selection, the false positive frequency ranged from 0.05 to 0.35%. The low false positive frequency indicates that we had set the threshold marker at an appropriate intensity level to exclude cells having low background autofluorescence. Cells transfected with pCEP4 (the expression vector containing no L1 element) or pL1RP(JM111)-EGFP (the expression vector containing a tagged L1 with a mutation known to eliminate retrotransposition) did not show a statistically significant difference in percent fluorescence above threshold, suggesting that either construct was an appropriate negative control for estimating the false positive frequency, presumably the result of autofluorescence, and strongly suggesting that events scored as positive represented authentic retrotransposition events and not recombination of a cDNA intermediate with genomic or episomal DNA.
EGFP was detectable by fluorescence microscopy in cells transfected with pCEP-EGFP as soon as 12 h post-transfection, indicating robust expression from the pCEP plasmid. After antibiotic selection and for the duration of the experiment, 94–100% of cells transfected with pCEP-EGFP were fluorescent above the threshold level, confirming sufficient antibiotic selection and indicating maintenance and expression of the pCEP4 plasmid.
The retrotransposition rate for each element tested was estimated by plotting the percent fluorescent cells exceeding the false positive frequency for each time point and calculating the slope of a line created by linear regression (Fig. A). In the hygromycin experiments, the estimated rate of retrotransposition for the L1RP element was 0.48% of transfected cells per day, while the estimated rate of retrotransposition of the L1.3 element was 3.4-fold lower at 0.14% of transfected cells per day.
Figure 3 (A) Percent fluorescent cells exceeding the false positive frequency was plotted as a function of time for the hygromycin experiments. Each time point represents cells analyzed from three independent transfection wells. The error bars indicate one standard (more ...)
In the puromycin experiments, the false positive frequency ranged from 0.02 to 0.26%. The retrotransposition rate for each element was estimated as in the hygromycin experiments described above (Fig. B). In the puromycin experiments, the estimated rate of retrotransposition for the L1RP element was 0.60% of transfected cells per day and the estimated rate of retrotransposition of the L1.3 element was 2.3-fold lower at 0.26% of transfected cells per day.