Figure 1.
Schematic representation of expression constructs for PBase variants, PBase fusion proteins, and the dual fluorescent UGm transposon used in this study.
(A) The mPB and NP-mPB coding sequences of mouse codon-optimized PBase were cloned into the pTriEx-HTNC plasmid by replacing the Cre cassette between SpeI and XhoI. The mPB and NP-mPB coding sequences were preceded by a hexa-histidine encoding sequence (6× His tag). NP-mPB included an additional nucleolus-predominant (NP) signal peptide. (B) The pTriEx-mPB-tGFP and pTriEx-NP-mPB-tGFP constructs expressed tGFP-fused PBases. The tGFP moiety allowed real-time imaging of the PBase variant subcellular distributions. The pTriEx-mPB-2A-eGFP and pTriEx-NP-mPB-2A-eGFP plasmids expressed PBase variants, which were linked to eGFP by the self-cleaving 2A peptide. All protein-encoding cassettes were transcriptionally regulated by a hybrid promoter composed of the CMV immediate early enhancer fused to the chicken β-actin promoter (CAG), and followed by a polyadenylation signal sequence (pA). (C) Flanking the 5′ and 3′ inverted terminal repeats (ITRs) (empty arrows), the dual fluorescent transposon (pXL-T3-Neo-UGm-cHS4X; UGm) carried a human ubiquitin C (UBC) promoter-driven H2B-eGFP-2A-mCherry-GPI (Gm) transgene that labeled the transposed cells with a characteristic chromatin EGFP and membrane mCherry dual fluorescence. Additional abbreviations: Neor, a neomycin phosphotransferase expression cassette providing resistance to G418 selection; 2× Ins, two copies of the insulator sequence from chicken β-globin.
Figure 2.
The NP-mPB showed a 3- to 4-fold increase in transposon integration efficiency.
The UGm transposon, alone or mixed with the mPB or NP-mPB expression vector, was co-electroporated into mouse or human embryonic stem (ES) cells and then selected for G418 resistance. Surviving colonies were stained by crystal violet and counted. There were more colonies in the NP-mPB group than in the mPB group in mouse ES (A), human ES (B), and Hela cells (C). (D–F) Transposition events were quantified by counting the colonies on culture plates. The transposition efficiency mediated by NP-mPB was increased 3- to 4-fold in mouse ES cells (D), 3-fold in human ES cells (E), and approximately 3-fold in Hela cells (F). n = 3 for each condition; bars indicate mean ± standard error; ** P<0.01, *** P<0.001.
Figure 3.
Evaluation of the transposition efficiency mediated by the gradient concentrations of PBase.
(A) The NP-mPB group demonstrated more colonies compared to the mPB group in HEK293T cells transfected with varying amounts of plasmid DNA. (B) Colony numbers were quantified, revealing a 2.5- to 6-fold increase in transposon integration efficiency for NP-mPB. The transposition efficiency was higher when more plasmid was transfected for both groups. No overproduction inhibition was noted. n = 3 for each condition; bars indicate mean ± standard error; ** P<0.01, *** P<0.001.
Table 1.
Insertion site analysis of NP-mPB assisted transposition events.
Figure 4.
The mPB and NP-mPB protein expression profiles in HEK293T cells transfected with pTriEx-mPB and pTriEx-NP-mPB plasmids.
(A) At 48 h after transfection, the transposase protein level in the pTriEx-mPB group was higher than that in the pTriEx-NP-mPB group. UGm plasmid was cotransfected as a control for transfection efficiency (anti-GFP). pTriEX-HTNC plasmid was transfected as a positive control for anti-His tag antibody (Cre). (B) Western blot analysis of nucleo-cytoplasmic separated lysates revealed that the mPB protein was distributed preferentially in the nucleus and the cytoplasm, whereas almost all NP-mPB protein was localized in the nucleus.
Figure 5.
Stability analysis of PBase protein, facilitated by flow cytometry.
Hela cells were transfected with pTriEx-mPB-2A-eGFP and pTriEx-NP-mPB-2A-eGFP plasmids. (A) APC fluorescence via anti-His tag antibody staining indicated the presence of mPB or NP-mPB. Because the eGFP moiety was cotranslated with PBase variants, the GFP fluorescence intensity was used as a reference for the mPB and NP-mPB translation levels of successfully transfected cells. (B and C) Y-axis represents accumulated cell counts. Although the NP-mPB group had a much smaller percentage of cells that were APC+ (4.65%) than the mPB group (18.2%), the percentages of GFP+ cells were similar (26.7% vs. 32.7%). (D) The APC fluorescence intensity in GFP+ populations represented the protein stability. The ratio of mean APC fluorescence (a.u.) of NP-mPB to mPB was 302 to 671.
Figure 6.
NP-mPB expression has little effect on cell viability.
To probe the potential cytotoxicity of mPB and NP-mPB expression, HEK293T cells were transfected with different amounts of the mPB, NP-mPB, mPB-tGFP, and NP-mPB-tGFP expression vectors. The relative cell viability was evaluated by the MTS assay. There was no significant difference among the different protein-expressing vectors. n = 3 for each condition; bars indicate mean ± standard error; P>0.05.
Figure 7.
Subcellular localization of mPB and NP-mPB.
Hela cells were transfected with mPB-tGFP or NP-mPB-tGFP expression vectors and immunostained with an anti-cyclin T1 antibody (red) and an anti-fibrillarin antibody (blue). (A) Fluorescence microscopy revealed that mPB was localized in the nucleus, but largely outside the nucleoli, as indicated by the fibrillarin. The cyclin T1 signal was dotted and spared the nucleus. (B) In contrast, NP-mPB was localized in the nucleus, with a strong staining in the nucleoli. There was a dotted pattern of NP-mPB in the nucleus beside the nucleoli. Some of the dots were overlapped with the cyclin T1 signal.