Table 1.
Plasmids used in this studya
| Plasmid | Description of plasmid |
|---|---|
| Driver plasmids | |
| pcDNA L1-RP (37) | Contains the 6.0-kb FL-L1 RP (49) cloned into pcDNA6 myc-his |
| pcDNA LRE4 | LRE4 was made by replacing the 6.0-kb L1-RP in pcDNA L1-RP with the 6.0-kb LRE4 NotI-AleI fragment from 99 LRE4 mEGFP (73) |
| pcDNA ORF1 (37) | Contains the 5′-UTR and ORF1 coding sequence from L1-RP cloned into pcDNA6 myc-his |
| pcDNA ORF2 (37) | Contains the 5′-UTR and ORF2 coding sequences from L1.3 (1, 21) cloned into pcDNA6 myc-his |
| pcDNA ORF2 (RT−) | Made by swapping a 0.8-kb XbaI-XbaI fragment containing the D702Y mutation (57) from pcDNA L1-RP (D702Y) (37) into pcDNA ORF2 |
| SVA H2D plasmids | |
| SVA H2D mneoI (37) | Contains 4.3-kb KpnI-NotI fragment consisting of SVA H2D and the mneoI retrotransposition indicator cassette cloned into pCEP-Pur |
| SVA H2D mEGFP (37) | Contains 4.8-kb KpnI-NotI fragment consisting of SVA H2D and the mEGFP retrotransposition indicator cassette cloned into pCEP-Pur |
| H2DΔAlu-like | SVA H2D contains 0.41-kb deletion from BlpI to PflMI |
| H2DΔVNTR1 | SVA H2D contains 1.13-kb deletion from PflMI to XcmI |
| H2DΔVNTR2 | SVA H2D contains a 1.31-kb deletion from, Tth111l to XcmI |
| H2DΔVNTR3 | SVA H2D contains a 0.76-kb deletion from, XcmI to XcmI |
| H2DΔSINE-R1 | SVA H2D contains a 0.12-kb deletion from PpuMI to AgeI |
| H2DΔSINE-R2 | SVA H2D contains a 0.32-kb deletion from BamHI to AgeI |
| H2D Hex only | SVA H2D contains 2.01-kb deletion from BlpI to AgeI |
| H2D Alu-like only | The Alu-like domain was PCR amplified using Phusion (NEB) as a KpnI/AgeI fragment using the following primers positioned from the BlpI to PflMI sites: Alu-likeForKpnI, 5′-TTTTTGGTACCGCTGAGCCAAAGCTGGACTGT-3′; Alu-likeRevAgeI, 5′-TTTTTTACCGGTCCAGACGATGGGCGGCCAGGC-3′ |
| H2D Hex-Alu | SVA H2D contains a 1.60-kb deletion from PflMI to AgeI. |
| H2DΔHex | Contains a 0.17-kb deletion from KpnI to BlpI. The hexamer was removed by digesting SVA H2D mEGFP with KpnI/BlpI followed by ligation with a phosphorylated double-stranded DNA oligonucleotide containing KpnI/BlpI sticky ends (KpnIOligo, 5′-CTTGC-3′; BlpIOligo, 5′-TCAGCAAGGTAC-3′) to restore the KpnI and BlpI sites and make H2DΔHex |
| SVA H11D plasmids | |
| pBS H11D | H11D was amplified as 1.7-kb KpnI-NotI PCR product with Phusion (NEB) using H11D_KpnIFor (5′-TTTTTGGTACCAGCAGAAGTGAGAAACCAGGCTCT-3′) and H11D_NotRev (TTTTTGCGGCCGCTTTGGTCTTCAGATGATTGCCAGT-3′) from the bacterial artificial chromosome used for the human genome reference sequence (RP11-465F2; obtained from BACPAC Resources Center [http://bacpac.chori.org/]). This SVA was identified because it differed at only 2 nucleotide positions from the SVA_D Alu-like consensus (81) (99.5% identity) and because it was short in length. Three independent PCRs were combined and sequenced by the Sanger method. SVA H11D differs at two positions in the SINE-R from the human reference genome. Both nucleotide changes are annotated as known SNPs (rs4331123 C → T and rs4554909 T → C). The combined PCR was digested with KpnI and NotI and subcloned into pBluescript KS(−) to make pBS H11D |
| SVA H11D mEGFP | SVA H11D was liberated from pBS SVAH11D as a 1.5-kb KpnI-PpuMI fragment, the EGFP cassette and last 0.1kb of the SINE-R was liberated as a 2.7-kb PpuMI-NotI fragment from pBS SVA2 mEGFP, and pCEP-Pur was digested with KpnI-NotI. The three fragments were ligated together to make SVA H11D mEGFP Pur |
| SVA H11D mneoI | SVA H11D mneoI Pur was cloned similarly to SVA H11D mEGFP Pur except using a PpuMI-NotI mneoI fragment from pBS SVA2 mneoI for a three-way ligation into pCEP-Pur |
| H11DΔAlu-like | SVA H11D contains a 0.43-kb deletion from NcoI to PflMI |
| H11DΔVNTR1 | SVA H11D contains a 0.56-kb deletion from PflMI to XcmI |
| H11DΔSINE-R1 | SVA H11D contains a 0.12-kb deletion from PpumI to AgeI |
| H11DΔSINE-R2 | SVA H11D contains a 0.32-kb deletion from BamHI to AgeI |
| H11D Hex-Alu | SVA H11D contains a 1.03-kb deletion from PflMI to AgeI |
| Other plasmids | |
| SVA SPTA mEGFP | To remove the 0.6-kb flanking DNA cloned with SRE-1, SRE1-mneoI was removed from pCEP SREI (37) as a 4.8-kb AleI-NotI fragment and subcloned into pBluescript at EcoRV-NotI sites to make pBS SPTA mneoI Δflank. To make SVA SPTA mEGFP, SVA SPTA was removed as a 2.5-kb KpnI-PpuMI fragment from pBS SPTA mneoI Δflank and swapped into pBS H2D mEGFP at KpnI-PpuMI. SVA SPTA now marked with mEGFP was removed as a 5.3-kb KpnI-NotI fragment and cloned into pCEP-Pur to make SVA SPTA mEGFP |
| 99 RPS mEGFP Pur (66) | Contains the full-length L1-RP (49) marked with the mEGFP retrotransposition indicator cassette in 99 Pur |
| 99 RPS JM111 mEGFP Pur (66) | Contains the full-length L1-RP (49) with amino acid substitutions (R261A/R262A) (61) in ORF1 marked with the mEGFP retrotransposition indicator cassette in 99 Pur |
| ORF1 mneoI (80) | Consists of the 5′-UTR and ORF1 coding sequence from L1.3 marked with the mneoI retrotransposition indicator cassette cloned into pCEP4 (Invitrogen) |
a
All drivers are cloned into pcDNA6 myc-his (Invitrogen). All elements marked with a retrotransposition indicator cassette are cloned into pCEP4 (Invitrogen) or modified pCEP4 backbones lacking the CMV promoter (99 backbone) or puromycin (pCEP-Pur and 99 Pur) instead of hygromycin resistance. For the SVA deletion constructs, most deletions were made by digesting pBS H2D mEGFP or pBS H11D mEGFP with both enzymes listed followed by blunting with T4 DNA polymerase (NEB) followed by religation. Each deletion marked with mEGFP was then flipped into pCEP-Pur as a KpnI/NotI fragment. The SVAs are cloned as KpnI-AgeI fragments, and the retrotransposition indicator cassettes are AgeI-NotI fragments.