Article Figures & Data
Figures
- FIG. 1.
The structure of plasmids used in this study. pNCT-tel contains a Neo gene, HSV-tk gene, and telomeric repeat sequences. pNCT-tel also contains an I-SceI recognition site, which is located between the Neo and HSV-tk genes. pNCT-tel was linearized with NotI prior to transfection. Linearization places the telomeric repeat sequences on one end to seed the formation of a new telomere following integration. pNTIL contains the Neo gene and an HSV-tk gene that contains a LoxP site in its 3′ untranslated region. pNTIL also contains an I-SceI recognition site, which is located at the 5′ end of the coding region of HSV-tk. pZTIL-tel contains a Zeo gene and a fragment of the noncoding region of the HSV-tk gene containing a LoxP site. pZTIL-tel was also linearized with NotI prior to transfection, both to facilitate targeted integration into the pNCT-tel plasmid and to place the telomeric repeat sequences on one end to seed the formation of a new telomere. pNTIL-100kb is similar to pNTIL, except that it contains a 4.5-kb fragment of the magmus gene (Target seq) for targeting to a site 100 kb from the telomere containing the pZTIL-tel plasmid. The magmus gene fragment is oriented with its distal end closest to the ampicillin gene (arrow).
- FIG. 2.
Southern blot analysis for identification of B3-Zeo clones in which the pZTIL-tel plasmid has been targeted to replace the pNCT-tel plasmid in clone B3. (a) The structure of the integrated plasmid sequences and the sizes of the predicted HindII restriction fragments are shown for the parental clone B3 and for clones containing the pZTIL-tel plasmid (B3-Zeo). (b) Southern blot analysis using the pNCTΔ plasmid of genomic DNA from clone B3 results in three HindIII restriction fragments (4.2, 2.3, and 2.0 kb) (arrows at left). B3-Zeo clones containing the targeted pZTIL-tel plasmid (clones 37, 59, and 95) contain two HindIII restriction fragments (4.2 and 1.8 kb; arrows at right). The location of the HindIII (H) restriction sites, the recognition site for the I-SceI endonuclease, and the recognition site for Cre recombinase (loxP) are shown. The molecular size markers (kb) consist of lambda bacteriophage HindIII fragments. This figure was generated by combining data from different Southern blots containing genomic DNA from B3-Zeo clones.
- FIG. 3.
The structure of the integrated plasmid sequences in the various cell clones used in this study. Clone B3-Zeo37 contains a single copy of the pZTIL-tel plasmid located adjacent to the telomere on the short arm of chromosome 16. Clone 10-7 was derived by targeted integration of the pNTIL-100kb plasmid to a site 100 kb from the telomeric pZTIL-tel plasmid in clone B3-Zeo37. The targeted integration of pNTIL-100kb resulted in the duplication of the target sequence on either side of the integrated plasmid, typical of insertion-type targeting vectors (56). The 10-7Cre clones were derived from 10-7 following Cre recombinase-mediated recombination between the LoxP sites, which resulted in the repositioning of the telomere to a location immediately adjacent to the pNTIL-100kb plasmid. The location of restriction sites and the size of restriction fragments are shown for the BglII (B), HindIII (H), NcoI (N), and XbaI (X) restriction enzymes. Also shown are the locations of the I-SceI and LoxP sites (boldface I and L, respectively) recognized by the I-SceI endonuclease and Cre recombinase, respectively. Symbols are shown for the location of the telomere, plasmid vector sequences (Amp/ori), Neo gene, HSV-tk gene, Zeo gene, and cellular sequence used for targeted integration of pNTIL-100kb (Target site).
- FIG. 4.
Southern blot analysis of the structure of the integrated plasmid sequences in clone 10-7 and the 10-7Cre clones. (a) Genomic DNA from the parental clone B3-Zeo37 and clone 10-h7 was digested with the BglII (B), HindIII (H), NcoI (N), and XbaI (X) restriction enzymes, and hybridization was performed using the pNCTΔ plasmid as a probe. The sizes of the restriction fragments in clone 10-7 correspond to those expected from targeted integration of the pNTIL-100kb plasmid into the site 100 kb from the telomere containing the pZTIL-tel plasmid (Fig. 3). Targeted integration in clone 10-7 was confirmed by rescue of the pNTIL-100kb plasmid and sequencing of the cellular DNA adjacent to the integration site. (b) Genomic DNA from clone B3-Zeo, clone 10-7, and seven of the 10-7Cre clones (Cre1 to Cre7) was digested with BglII or XbaI, and hybridization was performed using the pNCTΔ plasmid as a probe. The sizes of the restriction fragments in 10-7Cre1, 10-7Cre2, 10-7Cre3, and 10-7Cre6 correspond to those expected following Cre-mediated recombination between the LoxP sites in the pNTIL-100kb and pZTIL-tel plasmids (Fig. 3). Terminal restriction fragments that contain the telomeric repeat sequences are heterogeneous in length due to variability in the length of the telomere in different cells in the population (bracket, lower left). The sizes of the molecular size markers (kb) consisting of lambda bacteriophage HindIII fragments are shown.
- FIG. 5.
Decreased expression of transgenes following the relocation of the telomere to a site adjacent to the pNTIL-100kb plasmid. q-RT-PCR was used to determine the level of expression of the Neo gene and the HSV-tk gene in the parental clone 10-7 and four clones (10-7Cre1, 10-7Cre2, 10-7Cre3, and 10-7Cre6), that were derived from 10-7 following Cre-mediated recombination between LoxP sites in the pNTIL-100kb and pZTIL-tel plasmids. The results are given as percentage of the level of GAPDH gene expression. Error bars represent the standard deviation from three separate experiments. The levels of expression of both the Neo and HSV-tk genes were significantly reduced in all four Cre clones compared to clone 10-7 (P < 0.0001 for Neo and HSV-tk in Cre1, Cre 3, and Cre6; P < 0.0004 and P < 0.001 for Neo and HSV-tk, respectively, in Cre2).
- FIG. 6.
I-SceI-induced DSBs in clone 10-7A result in a high frequency of large deletions and terminal deletions. Genomic DNA from the parental clone 10-7A and eight of its Ganr subclones infected with the pQCXIP-ISceI retrovirus were analyzed by Southern blot analysis following digestion with the HindIII restriction enzyme and hybridization with the pNCTΔ plasmid as a probe. The locations of the three bands representing the pNTIL-100kb plasmid (gray arrows) and the two bands representing the pZTIL-tel plasmid (black arrows) are shown. Also shown are the sizes of the molecular size markers (kb) consisting of lambda bacteriophage HindIII fragments. Large deletions result in the loss of all three of the pNTIL-100kb-specific bands while terminal deletions result in the loss of the pZTIL-tel-specific bands.
- FIG. 7.
The frequency of small deletions resulting from DSBs at the I-SceI site in clones 10-7A and 10-7B. The frequency of small deletions determined by the ability of I-SceI endonuclease to cut a 1.8-kb PCR fragment spanning the I-SceI site in the integrated pNTIL-100kb plasmid. PCR was performed on genomic DNA isolated from pooled puror cultures of 10-7A and 10-7B 10 days after infection with either the pQCXIP control retrovirus (10-7A and 10-7B) or the pQCXIP-ISceI retrovirus (10-7AI and 10-7BI). The PCR product was then digested with I-SceI endonuclease, run on agarose gels, and stained with ethidium bromide, and the digital images were quantified by ImageJ analysis. The frequency of small deletions was then determined by dividing the intensity of the undigested band by the combined intensities of the digested and undigested bands.
- FIG. 8.
The absence of chromosome healing in Ganr/G418r subclones of 10-7. (a) The structure of the integrated plasmid sequences and the sizes of the predicted BamHI restriction fragments are shown for the parental clone 10-7 and for subclones with chromosome healing at the I-SceI site. (b) Genomic DNA from the parental clone 10-7A and eight of its Ganr/G418r subclones was digested with the BamHI restriction enzyme, and Southern blot analysis was performed using the pNCTΔ plasmid as a probe. Terminal deletions are evident by the loss of the 6.0-kb fragment containing the pZTILtel plasmid. The absence of chromosome healing is evident by the lack of diffuse bands (Fig. 4b) that are approximately 5 to 7.5 kb in length (bracket). Instead, there are discrete bands that vary in size, typical of rearrangements resulting from GCRs. The locations of the molecular size markers (kb) that consist of lambda bacteriophage HindIII fragments are shown.
Tables
- TABLE 1.
The frequency of large deletions and terminal deletions in Ganr subclones of 10-7A and 10-7B
Clone No. of large deletions (%)a No. of terminal deletions (%)b Total With large deletions Without large deletions 10-7A 14 (35%) 19 (47.5%) 14 (35%) 5 (12.5%) 10-7B 17 (43.6%) 21 (53.8%) 18 (46.1%) 3 (7.7%) - TABLE 2.
Percentage of cells in the population that contain small deletions at the I-SceI site
Clone Characteristic of cell population (% of cells) With uncut DNA Ganr With small deletionsa 10-7A 31.2 36 20.0 10-7B 32.5 54 15.0 ↵ a The percentage of cells with small deletions is determined by as follows: fraction of uncut PCR product × (1 − fraction of Ganr cells).
