Transgene expression, but not gene delivery, is improved by adhesion-assisted lipofection of hematopoietic cells

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Abstract

In contrast to adherent cells, cells growing in suspension and particularly hematopoietic cells, are notoriously difficult to transfect in vitro using nonviral approaches. In the present study, the effect of cell adhesion on gene transfer efficacy was investigated by allowing hematopoietic cells to bind to an adherent cell monolayer (ACM) before being subjected to cationic liposome-mediated DNA transfer. Human CD34 and T CD4 cell lines were cultivated on an ACM constituted of murine fibroblast NIH3T3 cells and transfected with a plasmid carrying the β-galactosidase gene. X-gal staining showed that up to 27% of the cells expressed the transgene. In contrast, less than 0.1% of these cells were positively transfected in suspension. This adhesion-assisted lipofection (AAL) procedure was also successfully tested on blood lymphocytes, since it resulted in up to 30% of transfected human primary T lymphocytes. Flow cytometry analysis performed on T lymphocyte subsets revealed that 8 and 9%, respectively, of CD4 and CD8 cells could be transfected with a plasmid carrying the green fluorescent protein gene. Other adherent cells, such as MS5 murine stromal cells or HeLa epithelial cells, were also a compatible matrix for AAL. Moreover, the pCMVβ plasmid was present in similar amounts in the nuclei of TF1 cells transfected in suspension or with the AAL procedure. These data raise the possibility that cell matrix/ hematopoietic cell interactions might govern expression of the transgene in hematopoietic cells growing usually in suspension, but not endocytosis of liposome/DNA particles and plasmid migration to the cell nucleus.

Introduction

At present, methods using viral vectors are the most promising for delivery and expression of foreign genes in vitro and in vivo. However, safety concerns and the difficulty in obtaining large quantities of recombinant viral vectors have prompted the search for efficient, nonimmunogenic and easy-to-prepare nonviral vector systems. Among them, cationic liposome-mediated gene delivery, which was pioneered by Felgner,1,2,3 has been demonstrated to be an effective approach presenting a low risk of intrinsic toxicity and pathogenicity.4 Cationic liposomes, which are easy to prepare on a large scale, form complexes with DNA through charge interactions in such a way that DNA is protected from degradative activities and virtually any size of plasmid molecule can be packaged. Liposome/DNA complexes bind to the negatively charged cell surface due to the presence of excess positive charges in the complex; the nature of nonspecific interactions results in the ability to drive expression of a foreign gene in host cells, including nondividing cells, with the greatest transfection efficiency obtained using adherent cells lines.5,6 However, many cell types, particularly primary cells, blood lymphocytes and more generally hematopoietic cells growing in suspension are largely resistant to this form of gene transfer,7 though successful transfection has been reported in mouse bone marrow cells.8

The sequence of cellular and molecular events underlying the complex phenomenon of DNA transfer remains highly speculative,9 but it is commonly accepted that endocytosis is the main mechanism of entry of liposome/DNA particles,10,11,12,13 and relatively high transfection efficiency is due to the intrinsic membrane-rupturing capability of cationic liposomes as a result of destabilizing the plasma and/or endosome membrane.14,15,16 In contrast to adherent cells, liposome/DNA particles have been reported to bind inefficiently to the cell surface of lymphocytes, suggesting that particles attach to membrane components involved in cell anchoring to the extracellular matrix and composed of cadherins or proteoglycans present on adherent cells only.6 This weak interaction between particles and membrane lymphocytes would prevent endocytosis of liposome/DNA particles and release of DNA into the cytosol of nonadherent cells. Because the ability of target cultured cells to be transfected with the liposome/DNA complex relies essentially on their adhesion capacity, we investigated the role of cell adhesion in gene delivery to hematopoietic cells. We primarily observed that hematopoietic cells could be transfected when cultured on an adherent cell matrix before being subjected to liposome-mediated DNA delivery. We then studied the usefulness of this new gene transfer approach in more detail using several types of established hematopoietic cell lines and primary lymphocytes of human origin, as well as different cell matrices. The role of cell adhesion in gene transfer optimization was also investigated.

Results

Coculture with adherent cells makes TF1 cells competent for gene transfer

Human TF1 cells, which grow in suspension, were loaded on to an NIH3T3 ACM as described in Materials and methods. The whole mixed cell population was then subjected to liposome-mediated DNA transfer using lipofectamine/pCMV β complexes. Two days after transfection, adherent cells were eliminated by adhesion on plastic flasks and expression of the bacterial β-galactosidase gene was assessed in purified TF1 cells and in pure TF1 cells transfected in suspension. Counting of dark blue-stained cells revealed that up to 26% of TF1 purified cells expressed β-galactosidase activity transferred with the pCMVβ vector. In contrast, less than 0.1% of blue cells was observed when TF1 cells were transfected in suspension (Figure 1). Total RNA was purified from cells harvested 48 h after transfection and β-galactosidase transcripts were subjected to a non-quantitative RT-PCR. Bacterial β-galactosidase mRNA level in TF1 cells transfected with AAL was 50 to 80% of what was found in NIH3T3 cells; transgene transcripts were poorly detected in TF1 cells transfected in suspension (Figure 2). Thus, it is obvious that culture of TF1 cells on a NIH3T3 monolayer authorizes a substantial improvement in liposome-mediated gene transfer.

Figure 1
figure1

Analysis of β-galactosidase expression in purified TF1 cells after transfection with lipofectamine/pCMVβ complexes. TF1 cells were transfected in suspension (a) or using the adhesion-assisted lipofection procedure on to a NIH3T3 cell matrix (b). Forty-eight hours after gene delivery, the purified TF1 cells were stained using X-gal.

Figure 2
figure2

Analysis of β-galactosidase transcripts in cells transfected with liposome/pCMVβ complexes. Cells were transfected using the AAL procedure or a standard lipofection (SdL) assay. Total RNA was purified from 5 × 105 transfected cells, subjected to DNase I and amplified by RT-PCR. PCR products encoding β-galactosidase DNA sequences were then electrophoresed, transferred to nitrocellulose and hybridized with a 32P-labeled specific DNA probe. RNAs from untransfected cells and RNAs subjected to both DNase 1 and RNase A did not show any RT-PCR products.

Purified TF1 cells transfected with AAL are essentially free of contaminating adherent cells

The purpose of these experiments was to assess the efficiency of the three 2-h adhesion steps used at the end of the AAL procedure. In practice, the purity of the cell suspension was evaluated by detection of specific cell surface markers. As shown in Figure 3a, less than 1.1% of the purified AAL transfected cells could be recognized by the anti-mouse mAb. In other respects, respectively 97.1% and 91.9% of purified transfected cells were detected with anti-HLA class I and anti-CD34 mAbs, compared with 99.5% and 94.6% for pure TF1 cells. The same transfection study performed using MS5 stromal cells as a matrix showed that virtually 100% of recovered suspension cells were recognized with both anti-HLA class I and anti-CD34 mAbs (Figure 3b). Taken together, these representative experiments show that NIH3T3 and MS5 contaminant cells are barely detectable in purified suspension cells and that essentially pure TF1 cells can be recovered after the three 2-h adhesion steps.

Figure 3
figure3

Purity of TF1 cells collected after the three 2-hour-adhesion steps. Purified CD34 TF1 cells transfected with adhesion-assisted lipofection performed with NIH3T3 (left) or MS5 (right) adherent matrix were examined by one-color flow cytometry. Murine MHC-H-2d antigen expressed at the surface of NIH3T3 cells was detected with an antimouse mAb to quantify the NIH3T3-contaminating cells. Human TF1 cells were identified both with anti-HLA class 1 and anti-CD34 mAbs. The second staining was achieved with FITC or R-PE-labeled goat antimouse F(ab′)2 fragment. The number shown in each quadrant indicates the corresponding percentage of cells with respect to the total number of analyzed cells. Displayed on the x axis is the fluorescence intensity on a logarithmic scale and on the y axis is the relative cell number. Pure NIH3T3 and TF1 transfected cells were used as a control.

AAL, but not adhesion, is optimized by prolonged coculture of TF1 cells with the cell matrix

In the interest of optimizing the AAL procedure, TF1 cells and NIH3T3 ACM were cocultured for increasing periods of time before being subjected to transfection. Purified TF1 cells were then assessed for bacterial β-galactosidase gene expression. As shown in Figure 4, the percentage of TF1 blue cells increased linearly for the first 6 to 8 h of coculture before reaching a plateau, but acquisition of the full competent state by TF1 cells required overnight coculture with the ACM before transfection. The ability of TF1 cells to bind to the ACM was studied as a function of the time to link the gene transfer efficiency to the adhesion/suspension state of TF1 cells. Surprisingly, 72% of TF1 cells were already bound to the cell matrix less than 2 h after being loaded on to the ACM. This indicates that prolonged coculture with the ACM is necessary for optimized liposome-mediated gene delivery and that adhesion of TF1 cells to the ACM precedes the acquisition of the features rendering TF1 cells competent.

Figure 4
figure4

Gene transfer efficacy in TF1 cells and adhesion of TF1 cells to the cell matrix as a function of time of suspension/adherent cell coculture. TF1 cells and NIH3T3 ACM were cocultured for increasing periods of time before being subjected to transfection. Purified TF1 cells were then assessed for bacterial β-galactosidase gene expression, as described in Materials and methods. The percentage of TF1 blue cells was plotted as a function of time of coculture before transfection. Adhesion of TF1 cells to the NIH3T3 ACM was also studied as a function of the time of cell coculture.

CD34 and CD4 cell lines are efficiently transfected with AAL

As a step towards examining primary cells, we examined the ability of the AAL procedure to mediate gene delivery in three different CD34 cell lines (TF1, KG1a, K562 cells) and in Jurkat CD4 T lymphocytes using NIH3T3, MS5 or HeLa ACMs; all suspension cells bound rapidly to the different ACMs. Two days after transfection, purified cells were examined for β-galactosidase activity. Pure CD34 cells and CD4 lymphocytes were also transfected in suspension. As expected, transfection performed with NIH3T3 ACM raised the percentage of blue-stained cells from less than 0.1% in the standard protocol up to 26%, 16% and 19% in TF1, KG1a and K562 cells (Figure 5). Lipofection of Jurkat cells on NIH3T3 ACM resulted in 18.5% transfection efficiency, as compared with 0.8% in controls, but the best experiments permitted β-galactosidase expression in up to 27% of the cells. AAL performed with the MS5 matrix gave essentially the same results as with NIH3T3 ACM, though CD4 cells were sometimes observed to be more permissive to DNA transfer with the MS5 matrix. Thus, up to 31% of Jurkat cells were transfected on the MS5 ACM. Use of HeLa matrix did not give such good data since only 4.9% and 3.5% of TF1 and KG1a cells turned blue. This nevertheless corresponded to strong enhancement of gene transfer efficacy compared with transfection of suspension cells; but the possibility that some blue cells counted as TF1 cells might be HeLa cells, cannot be rejected since these cells were more difficult to eliminate. Altogether, these results demonstrate that AAL permits an efficient transfection into a broad range of hematopoietic cell lines, and that many adherent cells can be used as a matrix.

Figure 5
figure5

Gene transfer to several lineages of human hematopoietic cells and to human primary lymphocytes. TF1, KG-1a, K562, Jurkat cells and human PBL were transfected with AAL using liposome/pCMVβ complexes and several cell matrices. Cells were transfected either in suspension or on to a NIH3T3, MS-5 or HeLa cell monolayer. Successful gene transfer was measured by X-gal staining as outlined in Materials and methods. Single asterisk and double asterisks indicate assays showing gene transfer efficiency of less than 0.1% or 0.03%, respectively. Transfection of K562, Jurkat and PBL on HeLa cell matrix cells was not done (nd).

PBL subjected to AAL are permissive to gene transfer

We next examined the performance of AAL in human PBL. Forty hours after transfection, PBL represented less than 15% of the whole mixed cell population, probably because primary lymphocytes were slowly or non-dividing cells. X-gal staining of purified lymphocytes revealed that up to 30% and 12% of PBL transfected on NIH3T3 or MS5 ACM expressed the transgene, in contrast to less than 0.1% for cells transfected in suspension (Figure 5).

Thus, the substantial enhancement of efficiency of DNA transfer into human PBL with the NIH3T3 and MS5 cell matrices was comparable to that observed above in hematopoietic cell lines, although primary lymphocytes were non-dividing cells.

EGFP is expressed in primary CD8 and CD4 lymphocyte subsets

The purpose of this work was to evaluate the performance of AAL in primary T lymphocyte subsets, directly in the whole mixed target/matrix cell population without the requirement for the three 2-h adhesion step. As described above, PBL were loaded on an NIH3T3 ACM before being transfected with liposome/pEGFP complexes. At day 2, cells were incubated with anti-CD4 or anti-CD8 mAbs. Cell labeling with R-PE-labeled anti-mouse Fab fragment enabled us to thoroughly distinguish CD4 and CD8 lymphocyte subsets from murine cells. The mixed NIH3T3-PBL cell population was then analyzed by two-colour flow cytometry for EGFP in lymphocytes carrying either the CD4 or the CD8 cell surface marker. Scoring for cells expressing the EGFP protein revealed that 8% and 9%, respectively, of CD4 and CD8 lymphocytes were fluorescent cells (Figure 6). Controls performed on cells transfected in suspension showed no significant EGFP fluorescence.

Figure 6
figure6

EGFP expression in CD4 and CD8 lymphocyte subsets. PBL were transfected on a NIH3T3 monolayer with plasmid pCMVβ or pEGFP. The mixed NIH3T3/PBL cell population was analyzed by flow cytometry two days after transfection for EGFP expression in CD4 and CD8 lymphocyte subsets. The gates corresponding to CD4 or CD8 negative cells have been cleared from the Figures. Plasmid pCMVβ was used to distinguish positive cells expressing EGFP from negative cells and background fluorescence.

Plasmid molecules enter the cells whether transfected in suspension or with AAL

A search for pCMVβ DNA sequences was undertaken in an attempt to follow the migration of plasmid molecules through the cell membrane and to the nucleus of transfected TF1 cells. Great care was taken in the isolation of clean and unruptured nuclei free from residual plasmid not contributing to gene expression and which might be trapped in the cell membrane or cytoplasm. Total plasmid DNA contained in transfected cell nuclei, whether integrated into cellular DNA or not, were then subjected to a Southern evaluation procedure enabling quantification of the β-galactosidase gene.

A study performed 48 h after transfection revealed that purified nuclei of TF1 cells transfected either in suspension or on a cell matrix contained a comparable copy number of plasmid molecules, ie about 200 plasmid molecules per nucleus, while 200 to 350 plasmid copies were found in the nucleus of NIH3T3 cells (Figure 7). One week after transfection, a mean of 8 pCMVβ plasmid molecules was detected in nuclei of transfected cells while transfection efficiency was considerably affected, 5 to 10 times less than at day 2, in terms of number of cells expressing the transgene; the rapid decrease of the transgene in transfected cells is consistent with what has been reported previously for transient transfection. Most of the plasmid molecules were found in the nucleus rather than in the cytosolic compartment of transfected cells. Because it is well-known that cell lysis with NP-40 leads to nuclei without an outer membrane, it can be hypothesized that detected pCMVβ DNA molecules were localized inside the nucleus rather than bound to the outer nuclear membrane of transfected cells. It can be concluded from these data that liposome particles containing plasmid DNA molecules enter the TF1 cells by endocytosis and are then conveyed to the nucleus whether these cells are cultivated in suspension or on a cell monolayer.

Figure 7
figure7

Southern blot analysis of pCMVβ DNA in transfected cell nuclei. Cells were transfected using the AAL procedure or a standard (SdL) lipofection assay. At days 2 and 7 after transfection, total nucleic acids were extracted from 5 × 105 purified transfected cell nuclei and digested by EcoRV and NotI. For Southern analysis, one tenth of digestion products were electrophoresed in 1.0% agarose gel, transferred to nitrocellulose and hybridized with a 32P-labeled β-galactosidase DNA probe. The hybridization signal corresponding to the 1.2 kb DNA fragment was quantified using a Phosphorimager and compared with serial dilutions of digested pCMVβ DNA. The evaluated number of pCMVβ plasmid molecules per nucleus of transfected cells was noted below the corresponding lane of every transfection assay (means ± s.d. of four or five determinations).

Discussion

In this report, we have demonstrated a transfection method termed adhesion-assisted lipofection which leads to a substantial improvement in liposome-mediated gene delivery in vitro into human hematopoietic cells. AAL achieved transfection with high efficiency not only of different cultured cell lineages such as CD34 cells and CD4 lymphocytes, but also of PBL, primary CD4 and CD8 lymphocyte subsets. This indicates that the AAL procedure is not restricted to established cell lines, but can also be utilized for primary cells. Moreover, we recently established that AAL was able to drive β-galactosidase gene expression in up to 27% of primary enriched hematopoietic stem cells from human cord blood (data not shown). Thus, AAL works independently of the target cells, since all cell types were transfected with the same range of efficiency, and is effective both in dividing and nondividing cells. In addition, this transfection procedure does not require a prior stimulation of the cells with a chemical agent.

Because of the unusual features of the cell matrix in improving the gene transfer, the possibility of a passive diffusion of the β-gal protein from the cell matrix to the human hematopoietic cells was explored. As shown in Figure 1, positive hematopoietic cells were strongly stained which corresponds to large amounts of protein in the cells. In contrast, passive diffusion of an exogenous protein into the TF1 cells would have produced weakly stained cells. Figure 4 shows assays in which TF1 cells were cocultivated 1 h to 14 h with NIH3T3 cells before transfection; in all these assays, most of the TF1 cells were attached to the cell matrix after a 2-h coculture and TF1 cells were intimately imbricated with NIH3T3 cells 2 days after transfection. Thus, in all assays, TF1 cells were grown and transfected in the same cell matrix environment and would have been comparably contaminated by released β-galactosidase in the event of protein diffusion from the cell matrix to hematopoietic cells. This was not the case because the number of blue TF1 cells did not depend on their coculture with the matrix after transfection but on the time of coculture before transfection. Moveover, purified TF1 cells transfected with AAL and overexpressing the β-gal protein were shown to contain similar amounts of the transgene transcript to those found in matrix cells, but it was barely detected in TF1 cells transfected in suspension. Finally, direct FACS analysis of TF1-NIH3T3 mixed cell population demonstrated that at least 14% of TF1-cells were recognized as expressing the EGFP protein when cells were transfected using the AAL procedure and pEGFP plasmid (data not shown).

Our results shed light on the possible implications and consequences of cell-to-cell adhesion on liposome/DNA complex routing and transgene expression. Our model, using the same cell either in a suspension or in an adhesion state allowed the question of the mechanism of liposome-mediated DNA transfer into adherent and nonadherent cells to be addressed. Transfection of several hematopoietic cell types growing in suspension was demonstrated here to be enhanced at least 200-fold when these cells were cultured on to an adherent cell monolayer. AAL efficiency performed with the MS5 matrix was comparable to that with NIH3T3 ACM for all types of hematopoietic cells tested. HeLa ACM increased gene delivery, but with less efficacy. Preliminary experiments performed with human endothelial cell matrix showed a transfer efficiency of about 3% both with TF1 and Jurkat cells. Thus, murine fibroblast cells and to a lesser extent, human epithelial-like and endothelial cells, shared features rendering human suspension cells permissive to liposome-mediated DNA transfer. On the other hand, it is probable that gene delivery into the different cell lines and into primary cells was regulated by a common pathway since all cell types were transfected with the same range of efficiency on a particular ACM. In other respects, studies investigating the role of stroma or extracellular matrix in retroviral infection revealed that the presence of a cell feeder layer during transduction protocols increases gene transfer efficiency.17,18,19,20 This enhanced transduction is probably due to the colocalization of retrovirus and target cells on specific domains of fibronectin molecules allowing hematopoietic cell transduction by exploiting unique ligand–receptor interactions.17,21,22 In this system, interactions between cells and fibronectin molecules were mediated via cell surface proteoglycan molecules, VLA-4 and/or VLA-5 integrins expressed at the surface of hematopoietic cells. The possibility that, in AAL, fibronectin or other cell-adhesion molecules carrying anionic residues and expressed at the surface of adherent cells only, might bind both hematopoietic cells and cationic liposomes, thus authorizing their colocalization and endocytosis of particles into the suspension cells, is not supported by our data. Indeed, the use of fibronectin-coated flasks failed to enhance the number of TF1 cells expressing the transgene significantly (data not shown). Additional experiments showed us that growth of TF1 cells in NIH3T3 culture medium supernatant, as well as culture of TF1 cells on anti-CD34 antibody-coated flasks, essentially failed to improve gene transfer (data not shown). In addition, the term ‘transfected in suspension’ is inaccurate because all TF1 cells attach strongly to the plastic when grown in serum-free medium and in the presence of liposome/DNA complexes. On the other hand, similar amounts of plasmid molecules were found to be conveyed to the nucleus of TF1 cells transfected in suspension or cocultured on a cell matrix and to the nucleus of NIH3T3 transfected cells. Altogether, these data show that adhesion of hematopoietic cells on culture flasks or a cell monolayer does not favor entry of plasmid into the cells. This also demonstrates that the plasma membrane, which has been described as the first critical barrier that must be penetrated in order to achieve highly efficient gene delivery into the cytoplasm of cells1,14,15 does not specifically prevent liposome/DNA complex delivery into hematopoietic cells, but probably acts as in adherent cells.

This article reports that adhesion of suspension cells to the ACM appeared clearly to precede, but not to accompany, the acquisition of the features allowing an optimized expression of the transgene. These results and evidence that plasmid molecules are equally conveyed to the cell nucleus whether the cells are cultivated in suspension or on a cell matrix support the hypothesis that suspension-cell-to-adherent-cell adhesion might be necessary but that further cell interactions with the ACM after adhesion are required for an efficient expression of the transgene; these cell interactions might govern the transgene expression by stimulation of cellular factors acting at the transcriptional level. The fact that DNA uptake and transgene expression do not always correlate in transfected cells, but that processes subsequent to DNA uptake could be even more important in determining gene expression, has been widely documented.16,23 Thus, delivery of plasmid DNA to cultured epithelial cells was demonstrated not to be a limiting factor, since virtually 100% of cells receiving a lipid–DNA conjugate rapidly transport exogenous DNA to the region of the cell nucleus but only a small portion of these cells, are able to express the DNA at detectable levels.24,25 We confirmed here that delivery of plasmid DNA into cultured cells would not be the only limiting factor, but would implicate features inherent in the cells as contributing to the poor efficiency of lipid-based gene expression observed in vitro. The putative cellular factors involved in expression of a transgene transferred with AAL could be compared with the regulators expressed by established stromal cells, which control the maintenance of hematopoietic reconstituting ability in human and murine stem cells.26,27,28,29

To date, most efforts have been made to improve the efficiency of DNA packaging and its delivery into the cell by development of new liposome formulations which have also to be compatible with administration to human subjects. It may now be necessary to direct some of our efforts toward understanding of the mechanisms which render cells responsive to DNA transfer.

Materials and methods

Cells

KG1a,30 K562 and TF131,32 are human hematopoietic cells expressing the CD34 cell surface marker. Jurkat is a human CD4 T lymphocyte. Both human CD34 and CD4 cell lines are grown in suspension. NIH3T3 and HeLa are respectively a murine fibroblast and a human epithelial-like cell line. MS5 is a murine stromal cell which has been reported to support the growth and maintenance of the hematopoietic reconstituting ability of murine and human blood progenitor cells.28,33,34 NIH3T3, MS5 and HeLa are adherent cells. Growth factor dependent TF1 cells were maintained with 10 ng/ml GM-CSF. Peripheral blood mononuclear cells (PBMC) were prepared from heparinized venous blood of healthy adult volunteers. Depletion of monocytes by two 30-min adhesion steps on a plastic flask resulted in less than 2% of the peripheral blood lymphocytes (PBL) expressing the monocytic CD14 marker at their surface.

Liposome

Lipofectamine reagent (GIBCO BRL, Eragny, France) used in these studies is a polycationic lipid composed of a positively charged lipid, DOSPA, and a neutral lipid, DOPE, in a 3:1 molar ratio.

Plasmid

The pCMVβ plasmid (Clontech, Palo Alto, CA, USA) carrying a full-length E. coli β-galactosidase gene under control of the cytomegalovirus (CMV) immediate–early promoter and enhancer was used to assess transgene expression in most of our studies. pEGFP-N3 (Clontech) carrying the enhance green fluorescent protein (EGFP) was used specifically for study of gene delivery into whole PBL and T-lymphocyte subsets. Plasmids were purified with the Qiagen EndoFree Plasmid Kit (Coger, Paris, France).

Adhesion-assisted lipofection (AAL)

Adherent cells (105) were plated in 1 ml of complete Dulbecco medium per well of a 24-well dish (TTP, ATGC Biotechnologie, Noisy le Grand, France) and grown for 6 h to approximately 70% confluency to form the adherent cell monolayer (ACM). At that time, 1.25 × 105 cells (TF1, K562, KG1a, Jurkat cells) or 2.0 × 105 primary lymphocytes in 1 ml RPMI medium were loaded on to the ACM; unless otherwise stated, the mixed cells were cocultivated overnight to allow the suspension cells to adhere to the ACM. According to the manufacturer’s protocol, 0.5 μg of plasmid pCMVβ in 25 μl of serum-free OptiMEM medium was gently mixed with 4 μg of lipofectamine in 25 μl of serum-free OptiMEM medium in a polystyrene tube and allowed to sit on the bench for at least 20 min to form complexes. The liposome/DNA complex was then diluted to 250 μl with serum-free OptiMEM medium. Just before transfection, cell culture supernatant was gently removed and cells were rinsed once with serum-free medium before being overlaid with 250 μl of the liposome/DNA complex. After 5-h incubation at 37°C, liposome/DNA complexes were aspirated and 1 ml of complete fresh medium (50% Dulbecco, 50% RPMI) was added. The cells were maintained at 37°C for 40 h until purification of the suspension cells and β-galactosidase assay. Serum and antibiotics were not present during lipid-mediated transfection.

Purification of nonadherent transfected cells

At 40 h after transfection, mixed cells were washed gently once with phosphate-buffered saline (PBS), harvested by trypsinization, resuspended in 2.5 ml of complete medium and incubated for 2 h at 37°C in a 25-cm2 flask to allow the adherent cells to attach to the bottom of the flask. The suspension cells were then gently collected, loaded into a well of a six-well dish and incubated for 2 additional hours as before. This last procedure was repeated once. The purity of the resulting cell suspension was estimated by FACS analysis.

X-gal staining procedure

X-gal staining was performed according to the modified standard procedure.35,36,37 Quantification of the transfected cells was performed by counting in randomly selected fields the number of blue-stained cells under inverted phase microscopy.

RT-PCR analysis

Total RNA was purified from 5 × 105 cells using the RNAPLUS (Quantum Bioprobe, Montreuil, France) procedure. The aqueous phase was extracted twice with chloroform-isoamyl-alcohol before isopropanol precipitation. RNA samples were resuspended in 20 μl of water. Two microliters of RNA were subjected to 1 unit of DNase I in a 20 μl reaction as described in the GibcoBRL kit. A 2 μl RNA sample was subjected to both DNase I and RNase A (1 μg/μl). DNase I was inactivated by the addition of 2 μl of 25 mM EDTA solution to the reaction mixture and heating for 10 min at 65°C. Half of the pretreated RNAs were retrotranscribed with SUPERSCRIPT II reverse transcriptase and 1 μg oligo-dt (GIBCO-BRL kit) in a 40 μl reaction supplemented with 3 mM MgCl2. Two microliters of single-stranded cDNAs were then subjected to 20 cycles of amplification essentially as described (Perkin Elmer Applied Biosystems, Courta- boeuf, France) using Lac1 (5′-CGGACTCTAGAGGAT CCGGTACT) and Lac2 (5′-CAGTTTGAGGGGACG ACGACAGT) oligonucleotide primers. Southern analysis of RT-PCR products was performed using a 32P-labeled DNA probe specific for the β-galactosidase gene.

Antibodies and reagents

34-1-2S mouse antimouse MHC-H-2d monoclonal antibody (mAb) (lgG2a)38 was kindly provided by Dr M Pla (Hôpital Saint-Louis, France). Mouse anti-HLA class I (lgG2a, HLA-A,B,C; MAS 1532, Harlan Sera-Lab, Sussex, UK), anti-CD34 (lgG1, QBEnd/10, Novo Castra, Newcastle, UK), anti-CD4 (lgG1, kappa; MT310, DAKO, Trappes, France), anti-CD8 (lgG1, kappa; M707, DAKO), lgG1 negative control (X-0931, DAKO) mAbs, as well as labeled goat antimouse mAbs (lgG H+L, Jackson Immuno Research Laboratories, West Grove, PA, USA) conjugated to fluorescein (FITC) or R-phycoerythrin (R-PE), were obtained from the manufacturers.

Flow cytometric analysis

Cells (2.5 × 105) were pelleted in a Falcon tube (2052) and resuspended in 100 μl PBS supplemented with 0.5% BSA. Cells were then incubated with specific mAbs for 20 min at 4°C followed by washing with 4 ml PBS-BSA. Washed cells were then incubated for 20 min at 4°C with FITC or R-PE-labeled goat anti-mouse F(ab′)2 fragment. Finally, the cells were washed once again with 4 ml PBS and resuspended in 0.5 ml PBS supplemented or not with 0.5 μg/ml propidium iodide. Flow cytometric analysis of stained cells was performed with a FACScan flow cytometer and a CellQuest 1.2 software (Becton Dickinson, Mountain View, CA, USA). Dead cells which stained with propidium iodide were excluded by prior electronic gating. Analysis of mixed NIH3T3-depleted PBMC transfected with liposome/pEGFP complexes was carried out by two-color flow cytometry to detect expression of EGFP protein in CD4 and CD8 T lymphocytes.

Harvesting cells and nuclei

Cells (5 × 105) were harvested 48 h or 7 days after transfection, washed once with PBS, resuspended in 500 μl washing buffer (140 mM NaCl, 1.5 mM MgCl2 10 mM Tris-HCI and 0.001% NP-40) and incubated for 5 min on ice. The cells were washed once again with 500 μl washing buffer. Washing with NP-40 detergent was performed to eliminate liposome particles and DNA plasmid molecules collected with culture medium supernatant or weakly trapped at the surface of the plasma membrane. Cells were then resuspended in 100 μl lysis buffer (3 mM CaCl2, 2 mM MgAc, 0.1 mM EDTA, 10 mM Tris-HCl pH 8.0, 1 mM DTT) supplemented with 0.2% NP-40 and incubated on ice for 10 min. A drop of the nuclei suspension was examined microscopically to ensure that the nuclei were not ruptured and cells were lysed completely. The nuclei solution was then gently layered on to 500 μl of a 0.32 M sucrose cushion in lysis buffer and centrifuged at 4°C at 400 g for 5 min. While the cytoplasmic fraction was discarded, the nuclei pellet was gently resuspended in 500 μl of lysis buffer and centrifuged once more.

Southern analysis of pCMVβ DNA content

Whole cells or nuclei were gently resuspended in 285 μl extraction buffer (10 mM Tris-HCl pH 7.5, 0.1 M NaCl, 10 mM EDTA). After addition of 15 μl SDS 10%, the samples were shaken vigorously for 1 min at room temperature and extracted with phenol/chloroform. Total nucleic acids were recovered by ethanol precipitation. Total nucleic acids were digested by EcoRV and NotI, electrophoresed in 1.0% agarose gel, transferred to nitrocellulose and hybridized with a 32P-labeled β-galactosidase DNA probe. The hybridization signal corresponding to the resulting 1.2 kb DNA fragment was quantified by phosphor-sensitive luminescence (Phosphorimager; Molecular Dynamics, Bondoufle, France) and compared with serial dilutions of digested pCMVβ DNA.

Acknowledgements

This work was supported by the Centre National de la Recherche Scientifique and the Institut Pasteur. We thank Jerry Bram for critically reading the manuscript.

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Correspondence to P Froussard.

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Keywords

  • liposomes
  • cationic liposomes
  • adhesion-assisted lipofection
  • gene transfer
  • hematopoietic cells

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