Targeted insertion of a transposon into the Pax1 locus. (A) Introduction of a single SB transposon copy into the Pax1 locus by insertion-type homologous recombination. The open boxes indicate homologous sequences for recombination. An Hsp68 minimal promoter-LacZ cassette and tetO were inserted between IR/DRs of the transposon. (B) Southern blot analysis of the targeted ES cells. DNA from targeted and wild-type (WT) ES cells were double digested with EcoO65I and PacI and analyzed with two different probes. The locations of the probes are shown in panel A.

ChIP analyses of the transposon locus. Formaldehyde cross-linked chromatin from tTR (no. 1) and control (no. 1) ES clones cultured with DOX for 3 days to exclude tTR from tetO was immunoprecipitated without antibody (No Ab) or with anti-dimethyl H3-K9, anti-trimethyl H3-K9, anti-dimethyl H3-K4, or anti-acetyl H3 antibody. Fivefold serial dilutions of input and precipitated DNAs from tTR and control clones were used for semiquantitative PCR. Oct4 and Mage-a2 were used as controls for euchromatic and heterochromatic regions, respectively.

Effect of heterochromatin on the excision of the transposon by transiently transfected SB transposase. (A) Schematic representation of the excision assay and sensitivity of PCR conditions. (Left) SB transposase (pCMV-SB11) was transfected into tTR and control clones in the presence of DOX. After 48 h, feeder cells were removed, and the remaining cells were cultured for 3 days on a gelatin-coated dish; genomic DNA was extracted, and nested PCR was performed. The excised transposon locus yielded a 498-bp PCR product. Arrowheads, primers for nested PCR; asterisk, excision footprint. (Right) A single copy of the excised DNA could be detected by this nested-PCR condition. An excision-positive ES clone was obtained, and genomic DNA was prepared. The indicated copy numbers of excision-positive genomic DNAs were mixed with 100 ng of excision-negative genomic DNA. (B) High excision frequencies of the tTR-positive clones at the defined genomic locus. (Left) Genomic DNA from each clone (1, 10, and 100 ng) was used as a template for nested PCR. Ten PCR amplifications were carried out for each clone. Note that about 100-fold-higher frequency of excision events was detected in tTR-positive clones than in control clones. Two independent experiments were performed, and a representative result is presented. M, molecular marker. (Right) Control experiment to evaluate the quality of isolated DNA. The Fas ligand locus was amplified by single-round PCR. (C) Expression of SB transposase after transfection. tTR and control clones were transfected with the SB11 transposase expression vector. The Western blot analysis after 48 h posttransfection is shown. Tubulin was used as a loading control.

Recruitment of SB transposase to a tTR-induced heterochromatic region. ES cells expressing tTR (tTR clone no. 1) and control ES cells (control clone no. 1) were transfected with SB11 transposase expression vector. After 48 h, the cells were subjected to a ChIP assay using anti-SB polyclonal antibody. Fivefold serial dilutions of input and precipitated DNAs from tTR and control clones were used for semiquantitative PCR. NRS, preimmune rabbit serum.

Schematic representation of the initial transposition reaction and localization of SB transposase. (A) Summary and schematic representation of steps occurring in the initial transposition event: step 1 (I), SB transposase binds to IR/DRs; step 2 (II), synaptic-complex formation, followed by excision and reinsertion of the transposon from the original donor site. IR/DR(R) and IR/DR(L) indicate right and left IR/DRs, respectively. (B) Colocalization of SB transposase with intense DAPI staining. After SB transposase gene transfection, ES cells were fixed, followed by staining them with anti-SB antisera (red) and counterstaining with DAPI (blue). Scale bar, 10 μm. (C) Intracellular distribution of SB and HP1 proteins. ES cells expressing exogenous SB10 or vector control were extracted with an NE-PER kit (see Materials and Methods), and the indicated fractions were examined by Western blot analysis using the indicated antibodies.

Schematic representation of tTR expression vector and characterization of stably expressed tTR cells. (A) tTR expression vector under the control of the CAG promoter and its control vector. Three individual clones generated by each vector were selected for further analyses. Hygro, hygromycin. (B) Sensitivity to G418. The tTR and control clones were cultured in G418-containing medium (150 μg/ml) for 6 days. Resistant colonies were stained with Giemsa solution. Clones expressing tTR completely perished, while all control clones survived. (C) X-Gal staining. X-Gal staining showed that tTR clones were negative while control clones were positive. These data suggest that the LacZ gene was completely silenced by tTR-induced heterochromatin.

Analysis of methylation status within the transposon. (Top) Detailed restriction map of methylation-sensitive restriction enzymes. The LacZ probe used is shown as a box under the LacZ coding region. (Bottom) Genomic DNA from tTR-positive clones was not digested with methylation-sensitive HpaII, indicating that the region was highly methylated. On the other hand, genomic DNA from control clones was completely digested. Genomic DNAs from tTR and control clones were digested with methylation-insensitive MspI (shown on the right). The tTR-positive and control clones were cultured with DOX for 3 days.

Analyses of reinsertion events in the presence of puromycin selection. (A) Experimental procedure to analyze for reinsertion events. After the transfection of SB transposase, tTR and control clones were inoculated into 96-well culture plates with the indicated cell numbers. Excision-positive cell populations were expanded, genomic DNA was isolated, and LM-PCR analyses were performed to identify reinsertion sites. (B) Screening for excision events. Excision-positive wells derived from 100-cell/well groups theoretically contained one excision event, and insertion sites were determined by LM-PCR. M, molecular marker. (C) Flanking transposon genomic sequences. The original donor site is located on chromosome 2, and reinsertion chromosomes are shown.