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. 2009 Apr 17;135(2):166-74.
doi: 10.1016/j.jconrel.2008.12.016. Epub 2009 Jan 12.

Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection

Richard N Cohen  1 , Marieke A E M van der Aa, Nichole Macaraeg, Ai Ping Lee, Francis C Szoka Jr
Affiliations

Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection

Richard N Cohen et al. J Control Release. .

Erratum in

  • J Control Release. 2009 Sep 1;138(2):185

Abstract

Nuclear uptake of plasmid DNA is one of the many cellular barriers that limit the efficiency of non-viral gene delivery systems. We have determined the number of plasmids that reach the nucleus of a transfected cell using an internally standardized quantitative PCR (qPCR) assay. We isolated nuclei using two different protocols: a density gradient technique and a detergent-based method. The density gradient procedure yielded nuclei with substantially less adhering plasmids on the outside of the nuclei. Using the density gradient protocol we determined that cells transfected with Lipofectamine lipoplexes or polyethylenimine polyplexes contained between 75 and 50,000 plasmids/nucleus, depending on the applied plasmid dose. Any increase above 3000 plasmids/nucleus resulted in only marginal increases in transgene expression. Furthermore, lipoplex-delivered plasmids were more efficiently expressed, on the basis of protein expression per plasmid number in the nucleus, than polyplex-delivered plasmids. This indicates that polymer may remain bound to some plasmids in the nucleus. Lastly, by sorting transfected cells into high- and low-expressing sub-populations, we observe that a sub-population of cells contain 3x greater plasmids/nucleus but express nearly 100x more transgene than other cells within a single transfection reaction. Taken together these results suggest the importance of considering the processes downstream from nuclear entry for strategies to improve the efficiency of gene transfer reagents.

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Figures

Fig. 1
Fig. 1. Nuclear isolation and quantitative PCR
(a) Diagram of the nuclear isolation methods used (see methods). (b) qPCR standard curves used for relative quantification of plasmids/nucleus. The threshold cycle (Ct) for various concentrations of plasmids containing either the luciferase sequence (pLuc), the genomic actin sequence (pAct) or the genomic gapdh sequence (pGap) were measured to calibrate the qPCR assay. The R2 value and the slope of the fitted linear regression line and the calculated PCR efficiency (E = 10(−1/slope) – 1) for each plasmid are shown. The difference between the Ct of pLuc and the Ct of pAct (ΔCtL/A) over the same range of concentrations is also plotted (inset). Error bars representing the standard deviation of triplicates in the qPCR assay are smaller than the symbols representing each data point.
Fig. 2
Fig. 2. Comparison of nuclear isolation methods
(a) Detected plasmids/nucleus using the PCR assay for nuclear DNA extracted from nuclei isolated from transfected cells using the indicated methods (Fig. 1a). *Indicates P<0.05 (Student’s t-test) in comparison to iodixanol nuclei from the same transfection conditions. For B16F10/PEI transfections, some isolated nuclei were treated with the restriction enzyme MfeI, which cuts within the luciferase sequence (but not actin or gapdh), before extraction of DNA and the resulting plasmids/nucleus values are indicated as the lower line in each bar. †Indicates P<0.05 (Student’s t-test) in comparison between digested and undigested nuclei. (b,c) Confocal microscope images of nuclei isolated from B16F10 cells transfected with rhodamine-PEI (red)/Cy5-pLuc (blue) complexes (which appear pink) using either the detergent method (b) or the iodixanol method (c) and stained with Sybr I (green). Scale bar = 10 μm.
Fig. 3
Fig. 3. Testing wash conditions to remove extranuclear DNA from plasmid- and polyplex-coated nuclei
(a) Confocal microscope image of nuclei isolated from untransfected B16F10 cells by the detergent method, incubated with Cy5-pLuc for 1 hour at 4°C and washed with lysis buffer. Scale bar = 10 μm. (b-e) Nuclei were coated with pLuc or PEI/pLuc polyplexes as in (a) but with non-fluorescent materials. (b) Detected plasmids/nucleus for pLuc plasmid-coated nuclei following the indicated nuclear washing steps. (c) Detected relative ratio of luciferase plasmid DNA to gapdh genomic DNA for pLuc plasmid-coated nuclei exposed to MlyI before or after DNA extraction. (d) Detected relative ratio of actin genomic DNA to gapdh genomic DNA for pLuc plasmid-coated nuclei exposed to MlyI before or after DNA extraction. (e) Detected relative ratio of luciferase plasmid DNA to gapdh genomic DNA for PEI/pLuc polyplex-coated nuclei exposed to MlyI before or after DNA extraction. Error bars represent standard deviation of triplicates in the qPCR assay.
Fig. 4
Fig. 4. Plasmids/nucleus and luciferase expression dose response of PEI polyplexes and LFN lipoplexes
B16F10 (a–b) or A549 (c–d) cells were transfected with PEI/pLuc polyplexes or LFN/pLuc lipoplexes at the indicated doses. (a,c) Detected plasmids/nucleus in nuclei isolated by the iodixanol method. (b,d) Detected luciferase expression in lysates from transfected cells using the Promega Luciferase Assay kit normalized to the amount of total protein determined by the Bradford assay. Error bars represent the standard deviation of triplicates in the qPCR assay or luciferase assay.
Fig. 5
Fig. 5. Measuring plasmids/nucleus in subpopulations of high- and low-expressing cells
B16F10 cells were co-transfected with 105 copies/cell each of PEI/pLuc and PEI/pAP (secreted alkaline phosphatase) polyplexes (a), PEI/pGFP only (b), no polyplexes (c), or PEI/pLuc and PEI/pGFP polyplexes (d). Trypsinized cells were then analyzed for GFP expression using a MoFlo fluorescence-activated cell sorting (FACS) machine. The GFP intensity was measured in the FL1 channel with the FL4 channel used as an internal autofluorescence control. Forward- and side-scatter analyses (not shown) were used to exclude cellular debris contamination of sorted cells. Cells co-transfected with PEI/pLuc and PEI/pGFP polyplexes (d) were sorted into groups of relatively low (e) and high GFP expression (f). Luciferase expression (g) and plasmids/nucleus (h) were then measured for each group. *Indicates P<0.05 (Student’s t-test) in comparison to transfected-unsorted cells. In (i) the data from (g) and (h) are re-plotted against each other. Error bars represent the standard deviation of triplicates in the qPCR assay or luciferase assay.
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