Determination of L1 retrotransposition kinetics in cultured cells

Abstract

L1 retrotransposons are autonomous retroelements that are active in the human and mouse genomes. Previously, we developed a cultured cell assay that uses a neomycin phosphotransferase ( neo ) retrotransposition cassette to determine relative retrotransposition frequencies among various L1 elements. Here, we describe a new retrotransposition assay that uses an enhanced green fluorescent protein (EGFP) retrotransposition cassette to determine retrotransposition kinetics in cultured cells. We show that retrotransposition is not detected in cultured cells during the first 48 h post-transfection, but then proceeds at a continuous high rate for at least 16 days. We also determine the relative retrotransposition rates of two similar human L1 retrotransposons, L1(RP)and L1.3. L1(RP)retrotransposed in the EGFP assay at a rate of approximately 0.5% of transfected cells/day, approximately 3-fold higher than the rate measured for L1.3. We conclude that the new assay detects near real time retrotransposition in a single cell and is sufficiently sensitive to differentiate retrotransposition rates among similar L1 elements. The EGFP assay exhibits improved speed and accuracy compared to the previous assay when used to determine relative retrotransposition frequencies. Furthermore, the EGFP cassette has an expanded range of experimental applications.

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), the EGFP gene disrupted by a sense orientation γ-globin intron (Intron) and the TK poly(A) signal (pA). The splice donor (SD) and splice acceptor (SA) sites of the intron are indicated. Cells only express EGFP when an L1 transcript containing the antisense EGFP marker undergoes splicing, reverse transcription and integration into chromosomal DNA. EGFP is then expressed from the pCMV promoter. (B) L1 elements tagged with the EGFP cassette are cloned into the pCEP-based mammalian expression vectors, which places a SV40 poly(A) signal (SV40pA) downstream of the EGFP-tagged L1. The pCEP-based vectors are able to replicate as episomes in HeLa cells by using the origin of replication for eukaryotic cells (OriP/EBNA). The vectors contain either the hygromycin or puromycin resistance genes. The pCEP-based constructs containing the tagged L1 elements were transfected into HeLa cells and antibiotic selection was begun 24 h post-transfection. After antibiotic selection was completed, the cells were analyzed by FACS. (C) Retrotransposition is detected by EGFP fluorescence under UV light. The cassette demonstrates near real time retrotransposition in a single cell as indicated in this photograph of fluorescent cells detected by fluorescence microscopy (400× total magnification) 3 days post-transfection with an EGFP-tagged L1_RP element.

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), the EGFP gene disrupted by a sense orientation γ-globin intron (Intron) and the TK poly(A) signal (pA). The splice donor (SD) and splice acceptor (SA) sites of the intron are indicated. Cells only express EGFP when an L1 transcript containing the antisense EGFP marker undergoes splicing, reverse transcription and integration into chromosomal DNA. EGFP is then expressed from the pCMV promoter. (B) L1 elements tagged with the EGFP cassette are cloned into the pCEP-based mammalian expression vectors, which places a SV40 poly(A) signal (SV40pA) downstream of the EGFP-tagged L1. The pCEP-based vectors are able to replicate as episomes in HeLa cells by using the origin of replication for eukaryotic cells (OriP/EBNA). The vectors contain either the hygromycin or puromycin resistance genes. The pCEP-based constructs containing the tagged L1 elements were transfected into HeLa cells and antibiotic selection was begun 24 h post-transfection. After antibiotic selection was completed, the cells were analyzed by FACS. (C) Retrotransposition is detected by EGFP fluorescence under UV light. The cassette demonstrates near real time retrotransposition in a single cell as indicated in this photograph of fluorescent cells detected by fluorescence microscopy (400× total magnification) 3 days post-transfection with an EGFP-tagged L1_RP element.

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. A 342 bp product diagnostic for loss of the intron was detected in each clone (lanes 5–10) and from a control plasmid template containing EGFP without the γ-globin intron (lane 3). Lane 2 contains the expected 1243 bp PCR product from a control plasmid containing EGFP with the γ-globin intron. Lane 4 contains DNA from untransfected HeLa cells and lane 1 contains no template as negative controls. A 1 kb molecular mass size ladder (Gibco BRL) is indicated in the flanking lanes (MW).

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 deviation in each direction. Sampling and analysis were performed at 8, 9, 11, 14, 16 and 18 days post-transfection. (B) Percent fluorescent cells exceeding the false positive frequency was plotted over time for the puromycin experiments. Each time point represents cells analyzed from four independent transfection wells. The error bars indicate one standard deviation in each direction. Sampling and analysis were performed at 3, 4, 5 and 6 days post-transfection. In both sets of experiments, retrotransposition rates were estimated from the slope of a line created by linear regression.