Regulated expression of a transgene introduced on an oriP/EBNA-1 PAC shuttle vector into human cells

<p>Abstract</p> <p>Background</p> <p>Sequencing of the human genome has led to most genes being available in BAC or PAC vectors. However, limited functional information has been assigned to most of these genes. Techniques for the manipulation and transfer of complete functional units on large DNA fragments into human cells are crucial for the analysis of complete genes in their natural genomic context. One limitation of the functional studies using these vectors is the low transfection frequency.</p> <p>Results</p> <p>We have constructed a shuttle vector, pPAC7, which contains both the <it>EBNA-1 </it>gene and <it>ori</it>P from the Epstein-Barr virus allowing stable maintenance of PAC clones in the nucleus of human cells. The pPAC7 vector also contains the <it>EGFP </it>reporter gene, which allows direct monitoring of the presence of PAC constructs in transfected cells, and the <it>Bsr</it>-cassette that allows highly efficient and rapid selection in mammalian cells by use of blasticidin. Positive selection for recombinant PAC clones is obtained in pPAC7 because the cloning sites are located within the SacBII gene. We show regulated expression of the <it>CDH3 </it>gene carried as a 132 kb genomic insert cloned into pPAC7, demonstrating that the pPAC7 vector can be used for functional studies of genes in their natural genomic context. Furthermore, the results from the transfection of a range of pPAC7 based constructs into two human cell lines suggest that the transfection efficiencies are not only dependent on construct size.</p> <p>Conclusion</p> <p>The shuttle vector pPAC7 can be used to transfer large genomic constructs into human cells. The genes transferred could potentially contain all long-range regulatory elements, including their endogenous regulatory promoters. Introduction of complete genes in PACs into human cells would potentially allow complementation assays to identify or verify the function of genes affecting cellular phenotypes.</p

Osteolysis induced by MOPC315.BM.Luc cells.

<p>Transmission X-rays of distal femurs of NSG mice injected with either RPMI 1640 (A) or MOPC315.BM.Luc (expressing a doxycycline inducible non-functional control shRNA) (B) show extensive osteolytic lesions in the latter (white arrows). Similarly, µCT 3D reconstruction of the respective NSG femurs demonstrate reduced wall thickness (white arrows) and trabecular structures in bone marrow infiltrated by MOPC315.BM.Luc (expressing a doxycycline inducible non-functional control shRNA) (D) compared to age-matched controls (C). (E–J) Quantification of bone structure changes in BALB/c mice 5–8 weeks after injection of MOPC315.BM.Luc (5×10⁵ cells) and in age-matched controls. (K) Measurement of Ca²⁺ in serum of BALB/c mice at various time points after injection with MOPC315.BM. *p<0.002.</p

Osteoclasts in bone marrow of NSG mice infiltrated with MOPC315.BM.Luc.

<p>(A,B) Representative images of TRAP+ staining from the bone marrow of an age-matched control NSG mouse injected with RPMI 1640. (C–E) Representative images of TRAP+ staining from the bone marrow of an NSG animal injected with MOPC315.BM.Luc. Inset in (C) shows bone marrow localization of transplanted MOPC315.BM.Luc cells using staining for IgA. TRAP+ osteoclasts are indicated by black arrow heads. In all cases scale bar is 200 µm. (F) Quantification of osteoclasts per mm bone surface. Controls are age-matched RPMI-injected NSG mice.</p

Delayed growth and BLI of Luciferase-transfected MOPC315.BM-cells.

<p>(A) Tumor take experiment with MOPC315.BM.Luc (2×10⁵ cells i.v.) (n = 13) was overlaid that of MOPC315.BM shown in Fig. 1B (also 2×10⁵ cells i.v.). The survival curves showing development of paraplegia are significantly different (p<0.0001). (B) Levels of M315 myeloma protein in sera of mice in (A). M315 was significantly different at day 10, 20 and 30 (p<0.002). N.D. = Not Detected. (C–E) All scales in photons/second/cm²/steradian. (C) Timeline of bioluminescent signals in a representative BALB/c mouse injected with 2.0×10⁵ MOPC315.BM.Luc i.v. (D) Examples of typical sites of affection. I: sternum, femurs, spleen. II, III: spine, femurs, spleen. IV: spine, femurs, spleen, shoulder. (E) Tumor growth of MOPC315.BM.Luc in four representative BALB/c mice pictured at day 28. (F) Luminescence emitted from typical sites of growth as a function of time (n = 13). MOPC315.BM.Luc mice (open boxes) are compared to non-injected control mice (filled boxes). *p<0.031 for femur and spine. **p<0.04 for all indicated sites. (G) Frequency of tumor growth in various sites on day 10, 21 and 31. *Signals from the shoulder region could not be attributed to particular bones. (H) Correlation of serum myeloma protein M315 measured on days 10, 21 and 32 and average luminescence [radiance (photons/second/cm²/steradian)] emitted in individual mice (r = 0.8929, p<0.001).</p

Flow cytometry and histology of MOPC315.BM DsRed cells injected i.v. in C.B-17 SCID (5×10⁶) and BALB/c mice (1×10⁶).

<p>(A) Upper left: Flow cytometric histograms of MOPC315.BM DsRed cells (solid line) and as a control MOPC315.BM cells (dashed line). Flow cytometry from a representative paraplegic C.B-17 SCID mouse. Upper right: Femur cells (solid line) and normal femur (dashed line). Lower right: spleen (solid line) and normal spleen (dashed line). The average and standard deviations for each organ are shown on each plot. Backgrounds in a non-injected mouse (to the right of the vertical line) were for femur 1.34% and spleen 1.68%. (B) I: H&E staining from a femur of a paraplegic BALB/c mouse. II: Fluorescence microscopy of sections of same femur. III: Femur from a mouse injected with non-fluorescent MOPC315.BM (control).</p

Co-localization of tumor cells and axial skeleton in MOPC315.BM.Luc injected in BALB/c nu/nu mice.

<p>(A) A photomontage of three representative BALB/c nu/nu mice (out of 14) serially imaged from day 0 to day 25. For days 0–8, the color scale was set at a radiance (photons/second/cm²/steradian) of 10⁴ to10⁵ (F stop 1, exposure 60 seconds or autoexposure 10⁴ counts, binning 8). From day 10 to 25 the scale was set at a radiance of 10⁵ to10⁶. (F stop 1, exposure 60 seconds or autoexposure 10⁴ counts, binning 1). (B, left picture) Ventral view of 3 mice injected 1h previously, showing weak signals emanating from the tibiofemoral regions. Such signals were consistently detected in 14/14 mice 1–2.5h after i.v. injection. Color scale was set at a radiance (photons/second/cm²/steradian) of 10⁴ to10⁵. (B, right picture) A close-up of the region of interest indicated by the red circle. (C) A close-up picture series of one mouse (day 33) showing typical sites of affection (skull, shoulder region, sternum, spine, femurs, tibia and spleen). The scale was set at a radiance of 10⁵ to10⁶ (F stop 1 autoexposure 10⁴ counts emission filter 620 nm). (D) A graph of the average luminescence [radiance (photons/second/cm²/steradian)] of BALB/c nu/nu male mice (n = 14) from day 0 to day 25. The luminescence value for each mouse is the average of ventral, dorsal and lateral (right/left) views. (E) A correlation plot of average luminescence for each mouse compared to their respective serum M315 values on day 10 (r = 0.9648, p<0.0001). Similar data were obtained on day 20. (F) DLIT and CT of a mouse with co-localization of tumor signal and the skeleton. The orange spheres depict where signal gradients are located. Pictures of various bones after removing skin or explanting organs, scales in photons/second/cm²/steradian, (G) spine, (H) ribs, (I) sternum, (J) femur and tibia, (K) kidneys, (L) lungs and heart, (M) spleen, (N) intestines and (O) liver.</p

Histology and immunostaining of organs from paraplegic BALB/c mice injected with 5×10⁵ MOPC315.BM (IgAλ2³¹⁵) cells i.v.

<p>(A) Bone marrow. Left: H&E staining. Right: Immunostaining with anti-λ or anti-κ (inset) antibodies, developed by HPRO (brown). (B) Spleen, red pulp. Left: H&E staining. Right: Immunostaining with anti-λ or anti-κ (inset) antibodies.</p