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Results: 4

Figure 4

Figure 4. From: Plug and play modular strategies for synthetic retrotransposons.

Schematic of iPCR mapping for L1 insertions. Multiple L1 insertions in the mouse genome are illustrated as solid green rods. One insertion is magnified to display its sequence features, including target site duplications (filled triangles) flanking the insertion and a poly(A) stretch at the 3’ end of the insertion. It should be noted that the majority of L1 insertions are variably 5’ truncated. Thus, the 3’ junction between an L1 insertion and its flanking genomic DNA is chosen to be the iPCR target. The relative position of both forward (Fwr, in red) and reverse (Rvs, in black) iPCR primers and restriction sites (M) are shown. Genomic DNA is represented by thin black lines and the PCR cloning vector as thicker blue line. Refer to text for details.

Wenfeng An, et al. Methods. ;49(3):227-235.
Figure 2

Figure 2. From: Plug and play modular strategies for synthetic retrotransposons.

SfiI-mediated swapping of reporter modules. (A) Efficient cassette exchange mediated by noncomplementary double SfiI sites. A double SfiI reporter cassette and an SfiI -ready recipient L1 plasmid were prepared by SfiI digestion, gel purified, ligated at a 1:5 vector:insert molar ratio, and used to transform E. coli strain TOP10 (Invitrogen). Plasmid DNA was prepared from each of twenty-eight colonies, digested with SfiI, and resolved on a 0.8% agarose gel in the presence of ethidium bromide. Colonies carrying a successful SfiI mediated cassette exchange event are predicted to produce two bands: 1.1 kb for the reporter cassette and 9.8 kb for the vector. M: 1 kb DNA ladder (New England Biolabs). (B) Directionality of SfiI mediated cassette exchange. The orientation of the double SfiI reporter cassette is checked by EcoRV digestion of all 19 recombinant clones (lanes 1–19). In all 19 cases, the band pattern is consistent with the orientation being correct. EcoRV is predicted to cut twice in a recombinant clone: once in the reporter insert and once in the backbone. If the orientation is correct, it should give a banding pattern of 9.6 kb and 1.3 kb. If the orientation is reversed, the bands should be 10.0 kb and 0.9 kb. A SfiI digested miniprep DNA (predicted to release 2 fragments of 9.8 kb and 1.1 kb, irrespective of orientation; lane 20) and a sample of uncut DNA (lane 21) serve as size controls. M: 2-log DNA ladder (New England Biolabs).

Wenfeng An, et al. Methods. ;49(3):227-235.
Figure 1

Figure 1. From: Plug and play modular strategies for synthetic retrotransposons.

Modular design of L1 vectors. (A) A schematic of the first generation of L1 vectors. Both the first cell culture assay vector and L1 transgene are housed in a pCEP4-based backbone as a NotI-BamHI fragment. The L1 element is under the control of either 5’UTR alone or two tandem promoters (a heterologous promoter “P” upstream of the NotI site, plus 5’UTR). (B) A blueprint for modularized L1 vectors. A hypothetical L1 vector can contain seven modules from 5’ to 3’ end: 5’ targeting homology arm, promoter, conditional control element, L1 coding sequence (ORF1 and ORF2), reporter, polyadenylation signal, and 3’ targeting homology arm. These modules are delimited by strategically placed restriction sites (indicated at the top of the vector) so that new vectors can be derived by swapping individual functional elements using flanking restriction sites. The same vector can also be used to generate vectors for different applications when subcloned into desired vector backbones. Selected restriction sites are denoted as “universal sites”, which should be uniformly preserved at specified positions in all modular vectors to facilitate module swapping. (C) Exemplary modular L1 vectors. pWA203 is an intermediate vector for targeting L1 into intron 3 of the human HPRT gene; ORFeusLSL is a conditional L1 transgene using Cre-loxP system; and pESD202 is our latest base vector in which the 3’ end fully confirms to the modular design shown in panel B.

Wenfeng An, et al. Methods. ;49(3):227-235.
Figure 3

Figure 3. From: Plug and play modular strategies for synthetic retrotransposons.

Retrotransposition assays with modular L1 vectors and an EBNA-1 mutant. (A) Schematic representation of assay vectors and results. All vectors shown are subcloned into pCEP-Puro backbone. pWA121, pWA122 and pTT014 are structurally identical except the following mutations: pWA122 carries D212G and D709Y point mutations that abolish ORF2 endonuclease and reverse transcriptase activities (marked by “x”); pTT014 has a frameshift mutation in the EBNA-1 gene on the vector backbone. pWA121 and pWA122 are pCEP-Puro version of previously reported pCEPsmL1 and pCEPsmL1mut. pTT014 is derived from pWA121 by introducing a 4 bp insertion at amino acid position 15 through AvrII restriction and filling in reactions. pTT011, pTT012, and pTT013 are modular ORFeus vectors in which the neoAI reporter was introduced by using the double SfiI scheme (dotted box). L1 expression is controlled by one or two tandem promoters, including CMV promoter (hatched box), 5’UTR of mouse L1 (open box) and/or CAG promoter (filled box). Cells are transfected in triplicate, selected with puromycin for 3 days, and for each transfection, three 10 cm dishes are seeded with 1 × 104 cells each under G418 selection for 9 days. Retrotransposition activity shown is the average number of colonies per 104 cells seeded. (B) Predicted open reading frames for the wt EBNA-1 gene (as in pWA121) and its frameshifted counterpart (as in pTT014). Note the frameshift mutation may produce a truncated 38 aa protein with the first 14 aa being in frame. The frameshift mutation is marked by a downward arrow. Drawn to scale. (C) Representative images of G418 resistant colonies from above assays.

Wenfeng An, et al. Methods. ;49(3):227-235.

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