1. Deoxyribonuclease I-like III Is an Inducible Macrophage Barrier to Liposomal Transfection 
Andrew Wilber, Michael Lu, Michael C. Schneider 
Molecular Therapy 
Volume 6, Issue 1, Pages (July 2002)
DOI: /mthe
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
DNASE-conditioned medium does not fully degrade liposome-associated plasmid. Shown is a representative experiment (n = 3) of ethidium stained 1% agarose electrophoresis of 2 μg free (-) or liposomal-associated (+ FuGENE 6) plasmid exposed to either control medium or medium conditioned by D1, D3, or D3(ΔCT). Incubation medium contained 10% fetal calf serum. After incubation for 1 hour at 37°C, we terminated nuclease activity with 50 μM EDTA, extracted DNA, and eluted in 40 μl water. Plasmid species are identified from top to bottom as nicked circular, linear, or supercoiled. Results show that neither D1 nor D3 completely degrades liposome-associated plasmid, thus the D3-BT effect cannot be solely explained by cell-free exposure. Liposome-associated plasmid exposed to D3-conditioned medium leaves mostly supercoiled plasmid protected, a result that does not differ from that seen with D3(ΔCT)-conditioned medium, which lacks barrier to transfection activity.
Molecular Therapy 2002 6, 35-42DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
D3-medium confers a barrier to liposomal transfection. (A) A representative anti-GFP immunoblot of N1-eGFP transfected HeLa cells exposed to medium conditioned by cells transfected with pcDNA3.1 vectors expressing either no insert (control, lane 1); D1 (lane 2); D3 (lane 3); D3(ΔCT) (lane 4); or D3(N196K) (lane 5). The westerns demonstrate that only the full-length, wild-type, D3 conditioned medium (lane 3) prevents gene transfection; expression of GFP by D1, D3(ΔCT), and D3(N196K) treated cells did not differ from that observed in controls. Immunoblots using anti-β-actin demonstrate near-equal loading of cell lysates. (B) Abundant GFP fluorescence in cells exposed to D1-, but not D3-, medium. Fluorescence in D1-exposed cells did not differ from control. (C) Anti-D3 immunoblots show similar levels of protein in cell lysates from D3, D3(ΔCT), and D3(N196K) expressing cells; both D3 and D3(N196K) express the expected 28-kDa D3 band. We did not observe the expected 2.2 kDa size shift for D3(ΔCT) protein. Actin immunoblots showed equal loading (data not shown).
Molecular Therapy 2002 6, 35-42DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Comparison of free DNA-nuclease activity and GFP-transfection efficiency. The bar graphs illustrate the free DNA-nuclease activity of conditioned medium (black bars) and the efficiency of GFP transduction of cells incubated in that medium (gray bars). Free DNA-nuclease activity was measured in units, one unit equaled 105 pixel volumes measured by densitometry of RDA results for 10 μl of sample. The mean activity by RDA of samples for D1, D3, D3(ΔCT), or D1-D3CT conditioned medium is shown. We measured GFP transfection efficiencies by the intra-experimental ratio of GFP expression in DNASE-conditioned medium relative to pcDNA3.1-medium exposed cells. Mean standard deviations are noted with bars. The cardinal result is that GFP expression is not detectable in cells lipofected with N1eGFP in D3-conditioned medium, but transfection efficiency was not significantly different than in control, D1, or D3(ΔCT)-medium, despite finding higher free DNA-nuclease activity in both D1 and D3(ΔCT)-media. D1-conditioned medium had significantly higher free DNA-nuclease activity relative to D3 medium (P < 0.001). Both D3(N196K) (null) and control media had similar negligible background free DNA-nuclease activity, and did not differ in GFP transfection efficiency. Finally, the transfection efficiency of GFP into HeLa cells was significantly decreased in medium conditioned by the chimeric D1-D3CT enzyme relative to control. Results are tabulated in Table 1. By visual inspection, GFP fluorescence results matched those expected by immunoblot.
Molecular Therapy 2002 6, 35-42DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Zymograms of mouse serum and medium conditioned by DNASE1, D3, and D3(ΔCT). A representative parallel zymogram result of 5 μl conditioned medium (2% fetal calf serum) for D1-transfected cells, 20 ml conditioned medium for D3- and D3(ΔCT)-transfected cells, and 5 μl mouse serum. D1 expression shows a band of glycosylated DNASE activity of 36–40 kDa, whereas the band of D3 and D3(ΔCT) activity in conditioned medium is smaller and sharper and electrophoresed at about ∼ 34 kDa in size. In most non-serum containing westerns, the bands expected for D3 run closer to 28 kDa. Most of the DNASE activity in murine serum zymograms is larger than 34 kDa, likely reflecting D1; however, in high serum conditions, protein migration is often aberrant, making the size determinations imprecise.
Molecular Therapy 2002 6, 35-42DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Immunoblots of murine serum, splenocytes, and tissues using polyclonal anti-D3 peptide antiserum. (A) This anti-D3 peptide immunoblot demonstrates the detection of D3 levels in 3 μl mouse serum as a 28-kDa band. A crescent of albumin is visible on top. (B) The anti-D3 antiserum detects the 34- and 28-kDa bands in immunoblots of adherent splenocytes (presumably macrophages), but only a 34-kDa band in non-adherent splenocytes. The actin blot below shows equal loading of protein. (C) We used the same antiserum against protein lysates from whole mouse organs, where it detects the expected 28-kDa bands in liver, thymus, and spleen. Liver also shows the uncleaved product (containing signal peptide) at 34 kDa. Bradford assay determined equal concentration (20 μg) of total protein loaded per lane. We detected only faint signals in lung. Pre-immune westerns failed to detect the D3 bands; and the bands were absent from a peptide-blocked western (data not shown).
Molecular Therapy 2002 6, 35-42DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
Studies of bone marrow derived macrophages and macrophage-conditioned medium. (A) Using anti-D3 antiserum, immunoblots of bone marrow-derived macrophage cell (Mϕ) lysates demonstrated the 34-kDa and 28-kDa bands. β-Actin immunoblots suggest total protein per lane is similar to that used in westerns of D3-transfected HeLa (Fig. 2C). (B) Top register illustrates RDAs of control (HeLa) and macrophages. We detect abundant free DNA-nuclease activity in macrophage-conditioned medium. Zymograms of these samples (10 are depicted below and show only a 28-kDa band, as expected for D3, in macrophage-conditioned medium. The levels of activity are comparable to those seen in medium conditioned by D3-transfected cells (Fig. 4). (C) The immunoblots of N1-eGFP transfected HeLa cells show that whereas control (HeLa-conditioned medium) allows GFP transduction, macrophage-conditioned medium blocks liposomal transfection of GFP into HeLa cells. Actin immunoblots below show equivalent protein loads. The results are evidence that bone-marrow-derived macrophages express D3, and a secreted DNASE activity consistent with D3 (at levels similar to those observed in our conditioned medium), and finally that macrophage-conditioned medium has barrier to transfection activity.
Molecular Therapy 2002 6, 35-42DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions