The effects of plasmid copy number and sequence context upon transfection efficiency

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The effects of plasmid copy number and sequence context upon transfection efficiency Wendy E. Walker, David J. Porteous, A. Christopher Boyd * Molecular Medicine Centre, Department of Medicine, University of Edinburgh, Western General Hospital, Crewe Road, EH4 2XU, UK Received 23 July 2003; accepted 10 October 2003

Abstract It is known that large P1 artificial chromosome (PAC) vectors exhibit reduced transfection efficiency in comparison to small plasmid vectors. We investigated the dynamics of this effect, by comparing expression from a small plasmid (4.7 kb) and a PAC vector (111 kb) containing the Enhanced Green Fluorescent Protein (EGFP) reporter gene under the control of a PCMV promoter. EGFP expression was detected by fluorescence activated cell sorting (FACS). We found that the lower transfection efficiency of PAC vectors represents both a smaller percentage of cells expressing the transgene, and a lower level of expression per cell. We have shown that the lower number of plasmid molecules administered per cell in a PAC transfection does not explain this effect, and that this effect does not act in trans. Surprisingly, dilution of a reporter construct with an irrelevant plasmid did not appear to compromise transfection efficiency; in fact, a dilution of 1/10 slightly enhanced transfection. Therefore, it seems that the plasmid content of a liposome – DNA complex need not be 100% reporter construct for optimum transfection efficiency. This discovery has potential practical utility in a number of applications. D 2003 Elsevier B.V. All rights reserved. Keywords: Plasmid; P1-artificial chromosome (PAC); Transfection; Size; Copy number

1. Introduction The delivery of DNA molecules to mammalian cells in order to direct expression of a transgene has a wide range of applications, but the mechanism of this process remains poorly understood. Transfection efficiency can be dissected into two components: the proportion of cells expressing a transgene and the level of expression per cell. Several factors will influence transfection, including the target cell type, the promoter, the delivery method (e.g. naked DNA

* Corresponding author. E-mail address: [email protected] (A.C. Boyd). 0168-3659/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2003.10.003

versus liposomal complex), the type of DNA vector chosen for delivery (e.g. small plasmid or PAC), and the amount of vector applied to the cells. For various reasons, we were interested in harnessing the cloning capacity of P1-artificial chromosome (PAC) vectors, in a general strategy to develop improved, physiologically regulated therapeutic transgenes [2]. It was important therefore to evaluate any detrimental effect of increased vector size upon conventional expression cassettes. Initially, we created a PAC containing an identical expression cassette to that in the small plasmid pEGFP-N (Clontech), to ascertain how PAC context affects expression [8]. The vector PACRC2cmvEGFP (Fig. 1) was created by introducing the PCMV-EGFP cassette derived from

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improved following dilution, suggesting a possible role for non-coding ‘carrier DNA’ as an agent to enhance transfection, a principal with a wide range of applications.

2. Materials and methods 2.1. Preparation of DNA

Fig. 1. Plasmid Schematic. (a) pEGFP-N (Clontech) and (b) PACRC2cmvEGFP both contain the same expression cassette: EGFP under the control of a PCMV promoter. (c) pUC18 and (d) PACRC2h do not contain the EGFP reporter gene. pEGFP-N and pUC18 are small plasmids, whereas PACRC2cmvEGFP and PACRC2h are large PAC vectors.

the pEGFP-N Clontech vector into a PAC vector, using a recombinogenic method [2]. This vector was transfected alongside the pEGFP-N vector into an immortalized cell line, in order to dissect the effects of PAC context upon expression. DNA was transfected into the COS-7 cell line using a lipid-integrin targeting peptide-DNA (LID) complex [4,5]. EGFP expression was detected with fluorescence activated cell sorting (FACS) and a dual-fluorescence dotplot analysis, described in Fig. 2. This method allows very sensitive detection of EGFP-expressing cells, and allows us to independently calculate the number of cells expressing EGFP and the average fluorescence of these cells, which is proportional to the level of EGFP expression [7]. In this paper, we show that expression from a large PAC vector is reduced in comparison to a small plasmid vector, but this effect is not the result of vector number and does not act in trans (e.g. upon a co-transfected DNA molecule). In addition, we show that it is possible to reduce the copy number of a reporter plasmid by dilution with an anonymous plasmid, without compromising its transfection efficiency. In fact, transfection efficiency may be slightly

A protocol was developed to optimize production of high quality PAC DNA; this protocol was also used in the preparation of small plasmids to ensure that all DNA preparations were of equal quality. DNA was first prepared with a Qiagen Maxiprep kit (cat. #12163, QIAGEN, Boundary Court, Gatwick Road, Crawley, West Sussex, UK). The manufacturers instructions were adhered to with the following modifications: a larger initial culture volume (200 ml per maxiprep column) was used to increase PAC DNA yield, 20 ml of P1, P2 and P3 buffers were added per 200 ml culture volume, and the maxiprep column was washed three times, instead of twice, with the QC buffer prior to DNA elution. Plasmid-Safe ATP-dependent DNase (cat. #E3101K, Epicentre Technologies, 726 Post Road, Madison, WI, USA) was used to digest any contaminating bacterial DNA in the preparation, according to the manufacturer’s instructions. A phenol/chloroform extraction and then a pure chloroform extraction were used to inactivate the Plasmid-Safe enzyme and remove protein and debris from the preparation. The DNA was then ethanol precipitated and resuspended in TE pH 8.0. PAC DNA was treated gently and never vortexed as this could easily shear the delicate molecule. DNA was stored at 4 jC. The vectors used for transfection are shown in Fig. 1. 2.2. Transfection The LID transfection system has been described by others [4,5]. LID transfection complex was prepared according to the method described by Dr. Steve Hart, Institute of Child Health, London. Briefly, 2.25 Al Lipofectin (GIBCO cat. #18297-011, Invitrogen, 3 Fountain Drive, Inchinnan Business Park, Paisley, UK) was combined with 120 Al P6 integrin-targeting peptide (100 ng/Al, a kind gift from Dr. Steve Hart)

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Fig. 2. FL1-H vs. FL2-H dotplot method. COS-7 cells were either (a) left untransfected or (b) transfected with the pEGFP-N vector, using the LID method. At 48-h post-transfection, FACS was performed. This figure shows the result of an FL1-H vs. FL2-H dotplot analysis. In the untransfected sample, all cells have an FL1-H (green fluorescence) reading approximately equal to their FL2-H (orange fluorescence) reading. However, in the pEGFP-N transfected sample, there is an additional population of cells delineated by region 1 (R1), where FL1-H>FL2-H. These appear to be cells expressing EGFP; this was confirmed by reverse transcriptase polymerase chain reaction (data not shown). (c) The percentage of cells in R1 and their average FL1-H fluorescence intensity were calculated. For untransfected samples, the percentage of cells in R1 is by definition 0, and the average fluorescence of the entire population was calculated instead as a measure of background fluorescence.

and 178 Al Optimem-1 (GIBCO, cat. #319-047, Invitrogen ) in a sterile 5-ml polystyrene tube. In a separate tube, 3 Ag DNA was diluted with Optimem-1 to make a final volume of 300 Al; this was then added to the Lipofectin/P6 mixture and the tube was shaken to mix. Finally, 900 Al Optimem-1 was added to make a final volume of 1.5 ml (this produced enough transfection complex for three wells of a 24-well plate). Where transfection incorporated a mixture of different DNA molecules, the DNA was mixed in Optimem-1 prior to addition of P6 and Lipofectin for complex formation, except where otherwise specified. COS-7 cells were obtained from ATCC and grown in DMEM (GIBCO cat. #41965-039, Invitrogen) + 10% Fetal Bovine Serum (GIBCO cat. #10106-151, Invitrogen) + 1% Penicillin/Streptomycin (Stock 10 000 U/ml, 10 000 Ag/ml, GIBCO, cat. #15140122). Cells were seeded in 24-well plates at a density of 2.5  104 cells per well. Transfection was performed 24-h later, when the cells had reached 60– 80% confluence. Each well of cells was washed with 2 ml

DMEM and then 500 Al of LID complex (containing 1 Ag DNA) was applied to the well. After 5 h, the transfection mixture was replaced with growth media. All transfections were performed in triplicate. 2.3. Fluorescence activated cell sorting (FACS) analysis At 48 h post-transfection, the cells were trypsinized, washed, and resuspended in PBS. EGFP expression was then analyzed with a Becton-Dickinson FACScan machine. The FCSpress software package was used to create FLH-1 vs. FLH-2 dotplots for each sample. Fig. 2 describes the dotplot method we used to quantify cells expressing EGFP and calculate their average green fluorescence intensity. Our method of analysis is thus similar to that reported by Blaauw et al. [1]. This two-dimensional dotplot method is more sensitive than a traditional histogram analysis, as it allows robust distinction of cells expressing very low levels of EGFP. A linear relationship has been dem-

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onstrated between a cell’s green fluorescence and the amount of EGFP protein in the cell [7]. Thus, the mean fluorescence of the transfected population will accurately reflect the mean level of EGFP expression per transfected cell. A two-tailed T-test was used to determine the significance of any differences in the percentage of cells transfected, or the average fluorescence intensity of transfected cells, between treatments. The p-values are reported in the text; a value of p V 0.05 was considered statistically significant.

3. Results A panel of vectors was assembled to investigate the effects of PAC context on expression (Fig. 1). The pEGFP-N (Clontech) and PACRC2cmvEGFP vectors both contain the EGFP gene under the control of a PCMV promoter. However, the former is a small plasmid (4.7 kb), while the latter is a large PAC (111 kb). The small plasmid pUC18 (Clontech) and the large PAC PACRC2h (a kind gift from Heather Davidson) were used as controls; these vectors do not contain EGFP. Preliminary experiments suggested that expression from a large PAC vector is reduced in comparison to expression from small plasmid. To investigate the components of this effect, we set up a series of transfections in the COS-7 cell line. 48 h after transfection, cells were analyzed by fluorescence activated cell sorting, with a dual dotplot analysis to detect expression (described in Fig. 2). The results of these studies are shown in Fig. 3. In the first experiment (Fig. 3a), COS-7 cells in a 24-well plate were transfected with 1 Ag of pEGFP-N or 1 Ag of PACRC2cmvEGFP per well. Transfection of the pEGFP-N vector produced 23% of cells expressing EGFP, with an average fluorescence intensity of 4586. In contrast, transfection of the PACRC2cmvEGFP vector produced only 4% of cells expressing EGFP with an average fluorescence intensity of 441. Thus, both the percentage of cells expressing EGFP ( p = 0.0036) and the average fluorescence intensity of these cells ( p = 0.0013) were significantly lower with the PAC vector, despite an identical expression cassette in these two vectors. Initially, we believed that this might be a copy number effect, due to using equal mass, rather than

molarity of the two DNA molecules (as the PACRC2cmvEGFP vector is 111 kb while pEGFP-N vector is only 4.7 kb, there will be approximately 24fold fewer copies of the PAC in 1 Ag). To test the effect of vector copy number, we mixed pEGFP-N plasmid with an anonymous DNA molecule: pUC18, which does not contain a mammalian expression cassette (Fig. 1). About 42.5 ng pEGFP-N was diluted with 957.5 ng pUC18 to produce the same EGFP transgene copy number and total DNA weight as in 1 Ag neat (undiluted) PACRC2cmvEGFP vector. This mixture was applied to a single well in a 24-well plate, in the first experiment, alongside 1 Ag pEGFPN, 1 Ag PACRC2cmvEGFP (as previously mentioned) and a negative control (1 Ag pUC18) (Fig. 3a). Surprisingly, the percentage of cells transfected (32%) and the average fluorescence of the transfected population (4907) following transfection of the pEGFP-N/pUC18 mixture were not reduced to the levels seen with 1 Ag PACRC2cmvEGFP; In fact, the percentage of cells transfected was slightly higher than that following transfection of 1 Ag pure pEGFP-N (this difference was statistically significant; p = 0.0338) (Fig. 3a). Thus, the low efficiency in PAC transfections does not appear to be a result of lower transgene number per se. One might imagine several ways in which large PAC vectors could affect transfection. For example, PAC DNA might have a gross effect, disturbing the transfection process (e.g. LID complex may not form effectively in the presence of the large PAC DNA). A second experiment was done to test whether PAC vectors are able to alter transfection efficiency in trans (e.g. of co-transfected DNA molecules). pEGFP-N was mixed with PACRC2h, which is identical to PACRC2cmvEGFP except that it contains a PCMVhalactosidase cassette in place of the PCMV-EGFP cassette (described in Fig. 1).About 42.5 ng pEGFPN was diluted with 957.5 ng PACRC2h to produce the same EGFP transgene copy number and total DNA weight as in 1 Ag of undiluted PACRC2cmvEGFP vector. This mixture was applied to a single well in a 24-well plate, alongside controls of 1 Ag pEGFP-N, 1 Ag PACRC2cmvEGFP and a negative control (1 Ag PACRC2h) (Fig. 3b). The percentage of cells transfected (39%) and the average fluorescence of the transfected population (4541) following transfection with the pEGFP-N/PACRC2h mixture were not re-

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Fig. 3. The effects of plasmid size and copy number upon expression. This figure shows the percentage of cells expressing EGFP and the average green fluorescence intensity of this population, as measured by FACS dotplot analysis (see Fig. 2) following transfection of the COS-7 cell line. (a) The effects of equalizing pEGFP-N and PACRC2cmvEGFP copy number. Cells were transfected with (1) 1 Ag pEGFP-N, (2) 1 Ag PACRC2cmvEGFP, (3) 42.5 ng pEGFP-N + 957.5 ng pUC18, or (4) 1 Ag pUC18 using the LID method, or (5) left untransfected. (b) The effects of diluting pEGFP-N with PACRC2h. Cells were transfected with (1) 1 Ag pEGFP-N (2) 1 Ag PACRC2cmvEGFP, (3) 42.5 ng pEGFP-N + 957.5 ng PACRC2h, or (4) 1 Ag PACRC2h using the LID method, or (5) left untransfected. (c) The effects of serially diluting pEGFP-N with pUC18. Cells were transfected with (1) 1 Ag pEGFP-N, (2) 100 ng pEGFP-N + 900 ng pUC18, (3) 10 ng pEGFP-N + 990 ng, (4) 1 ng pEGFP-N + 999 ng pUC18, (5) 100 pg pEGFP-N + 999.9 ng pUC18, or (6) 1 Ag pUC18 using the LID method, or (7) left untransfected. (d) Altering the complex in different ways. Cells were transfected with (1) 1 Ag pEGFP-N, (2) 100 ng pEGFP-N + 900 ng pUC18, mixed before complexing, (3) 100 ng pEGFP-N + 900 ng pUC18, complexed separately (4) 100 ng pEGFP-N + 0.75 Al Lipofectin + 4 Ag P6, (5) 100 ng pEGFP-N + 0.075 Al Lipofectin + 400 ng P6, or (6) 1 Ag pUC18, using the LID method, or (7) left untransfected. The DNA quantities listed were applied to single wells in 24-well plates.

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duced to the levels seen with pRC2cmvEGFP (5%, average intensity 752), but were comparable to the levels seen following transfection of 1 Ag undiluted pEGFP-N vector in this experiment (37%, average intensity 4542) (Fig. 3b). This suggests that the factors responsible for low transfection efficiency of PAC vectors do not act in trans upon a cotransfected DNA molecule. It seems, therefore, that the effect on PAC expression is purely cis-acting, rather than the result of a more gross alteration of the transfection process by the large PAC molecule. The fact that dilution of a plasmid did not reduce its transfection efficiency was in itself surprising and we decided to investigate this phenomenon further. Keeping the total amount of DNA constant at 1 Ag, pEGFP-N was serially diluted with pUC18 (from 1/10 to 1/10 000) and transfected into COS-7 cells using the LID method, alongside controls of 1 Ag undiluted pEGFP-N and 1 Ag pUC18. We found that serial dilution resulted in an initial increase in transfection efficiency, before a decline, i.e. undiluted DNA did not produce the optimum levels (Fig. 3c). Transfection of undiluted pEGFP-N vector produced only a moderate level of expression in this experiment (9% of cells transfected with an average intensity of 3774). Dilution of pEGFP-N 1/10 with pUC18 significantly increased the percentage of cells transfected (16%, p = 0.0244), without having a significant effect upon the average fluorescence intensity (3858). Dilution of pEGFP-N 1/100 with pUC18 produced a similar percentage of cells expressing EGFP (11%) and average fluorescence intensity of the transfected population (3025) to the undiluted pEGFP-N transfection. Even when pEGFP-N was diluted 1/10,000 with pUC18, EGFP expression could be detected (1% of cells transfected, with an average intensity of 2842). A similar pattern of transfection efficiency was seen when pEGFP-N was substituted with PACRC2cmvEGFP (dilution 1/10 with pUC18 gave the optimum transfection efficiency, results not shown) or a small plasmid expressing EGFP under a different promoter (results not shown). Finally, we tested whether the anonymous plasmid DNA had to be present in the same complex as the reporter plasmid to increase the transfection efficiency. 100 ng pEGFP-N and 900 ng pUC18 were either (1) mixed prior to formation of the LID complex, or (2) mixed with the appropriate amounts

of Lipofectin and P6 separately and complexed prior to applying the two complexes simultaneously to the cells. In order to rule out the possibility that it is merely a reduction in the amount of reporter plasmid, rather than the inclusion of an anonymous DNA molecule, which enhances transfection, two controls were included. In the first control, the amount of pEGFP-N in the complex was reduced without altering the quantities of the other LID components (e.g. to 100 ng pEGFP-N + 0.75 Al Lipofectin + 4 Ag P6 per well). In the second control, the entire LID complex was scaled down (e.g. 100 ng pEGFPN + 0.075 Al Lipofectin + 400 ng P6 per well). These mixtures were transfected into the COS-7 cell line alongside controls of 1 Ag pEGFP-N and 1 Ag pUC18 (Fig. 3d). Once again, mixing the pEGFP-N plasmid with pUC18 plasmid prior to complexing, increased expression (33% transfected, average intensity of 4627), in comparison to transfection of pure pEGFP-N plasmid (29%, average intensity 4544). When the plasmids were complexed separately and then applied to the cells simultaneously for transfection, 14% of cells were transfected, with an average intensity of 3909. This shows that it is necessary to mix anonymous DNA with the reporter plasmid before complex formation (so that the two plasmids are present in the same complex) in order to achieve an increase in transfection efficiency. Expression was also reduced in the two controls where the amount of reporter gene was reduced without the addition of an anonymous plasmid, either by just reducing the amount of DNA (12%, average intensity 3889), or by scaling down the entire complex (0.33%, average intensity 4168). This shows the increase was not simply an effect of lowering the transgene copy number.

4. Discussion This study demonstrates that in a transient transfection system, expression from large PAC vectors is reduced in comparison to that from small plasmids. This reduction represents both a smaller number of cells expressing the transgene and a lower level of expression per cell. We have shown that this is not attributable to the lower plasmid number, and is not a trans-acting effect.

Our findings also demonstrate that dilution of a reporter plasmid with an anonymous plasmid (e.g. pUC18) does not reduce transfection efficiency on a pro rata basis. Indeed, in some experiments (e.g. Fig. 3c and d), certain dilution factors enhanced transfection. We have recently observed similar effects of plasmid dilution and PAC transfection using alternative transfection reagents such as Saint-Mix (Synvolux Therapeutics) and Trojene (Avanti Polar Lipids), and when substituting a hgalactosidase reporter plasmid for pEGFP-N (results not shown). This suggests that the effects we have observed are not limited to the LID transfection system or the GFP reporter gene. It would be interesting to test the effects of mixing the reporter plasmid with other forms of DNA, such as CpG-methylated or mammalian genomic DNA. Additional dilutions between 1 Ag pure pEGFP-N and 10 ng pEGFP-N + 990 ng pUC18 should be performed to determine the optimum mixing ratio (which may vary between plasmids). Further work will be needed to clarify the basis of the decrease in transfection efficiency with PAC DNA and the effects of plasmid dilution. Crucially, the LID complexes should be characterized by techniques such as dynamic light scattering and electron microscopy, to determine whether proper complexes are being formed in the presence of PAC DNA, and whether complexes containing PAC DNA, a single plasmid, or a mixture of plasmids have similar physiochemical properties. In addition, the mechanism of the increase in expression should be investigated. Confocal microscopy studies using fluorescently labeled-DNA could be used to assess differences in DNA uptake/ trafficking with the different LID formulations. Plasmid microinjection could be used to determine whether PAC DNA and carrier DNA have an effect on expression following nuclear or cytoplasmic delivery. A complementary study by Kreiss et al. [6] compared the expression levels from a series of luciferase reporter constructs of increasing size. As in our study, expression declined as a function of vector size. This suggests that the size-effect is not specific to the EGFP reporter gene. It also demonstrates that this effect is not limited to very large PAC vectors, but also applies to differently sized traditional plasmid vectors. Furthermore, this study implies that this effect probably does not arise through a gross effect on lipoplex structure, as the

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lipoplexes had similar physiochemical properties regardless of plasmid size. Although we cannot yet explain this effect, there are a number of potential applications. It may be possible to transfect multiple reporter constructs simultaneously, to facilitate multiple measurements from a single sample, without compromising the transfection efficiency of the individual plasmids. This could be particularly useful to in vivo applications, where sample numbers may be limited. Some experiments require DNA of a certain quality and purity (for example, endotoxin-free DNA is often required for in vivo experiments). If the identity of the bulk of transgene DNA is not important, it may be useful to generate mass amounts of a common plasmid of this quality, which can subsequently be mixed with small quantities of the relevant plasmid for individual experiments. Non-coding ‘carrier’ DNA could be used to enhance transfection efficiency for in vitro experiments, and might even prove of therapeutic benefit in gene therapy applications. Plasmid dilution might increase the level of transgenic protein production. Finally, there is one report that suggests that plasmid mixing may have a similar beneficial effect in vivo. Braun et al. [3] performed a trial investigating the possibilities of mixing up to four plasmids encoding different viral membrane bound glycoproteins for DNA immunization: they found that that mixing the plasmids had very little effect upon the magnitude or bias of the immune response to the individual components.

5. Conclusion This study aimed to investigate how the DNA content of a liposomal complex affects transfection efficiency. We found that PAC context appears to compromise transfection efficiency: this effect is not attributable to copy number and is cis-acting. We also found that dilution of a reporter construct with an anonymous plasmid did not necessarily compromise transfection efficiency, and in some formulations actually enhanced it. These findings, and the results of others, point to the need for a better understanding of the molecular properties of transfection complexes and of the transfection process.

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Acknowledgements We would like to thank Dr. Steve Hart, Institute of Child health, London, for the kind gift of the P6 integrin-targeting peptide, and Heather Davidson and Ann Doherty for their helpful comments. This work was supported by an ORS scholarship from the CVCP (WW) and by the UK Medical Research Council (DP and ACB).

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[6]

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