Cationic liposome-mediated DNA transfection in organotypic explant cultures of the ventral mesencephalon

Abstract

We have examined the potential of cationic liposomes as a tool for approaches to gene therapy in the CNS. Our previous work has shown that cationic liposomes formulated from 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioleoyl-L-α-phosphatidylethanolamine (DOPE) could achieve high transfection levels in a neuronal cell line (McQuillin et al. Neuroreport 1997; 8: 1481–1484). We therefore wished to assess transfection efficiencies in organotypic cultures from the brain with a reporter plasmid expressing E. coli β-galactosidase in order to mimic an in vivo model. Explant cultures were generated according to the method of Stoppini et al (J Neurosci Meth 1991; 37: 173–182) with slight modifications. Brain slices were maintained on transparent porous membranes and were observed to maintain their intrinsic con- nectivity and cytoarchitecture to a large degree over periods of up to 6 weeks in culture. CNS tissue was obtained from rats at birth or 5 days after birth. After transfection β-galactosidase expression was detected in cells of both neuronal and non-neuronal morphology. Control cultures were exposed to liposome alone and a plasmid that had the β-galactosidase gene insert removed. Only low levels of endogenous β-galactosidase reactivity were seen in these control cultures. DC-Chol/DOPE-mediated transfection was confirmed using a RT-PCR protocol capable of differentiating between untranscribed plasmid DNA and RNA generated from the transfected vector. These results suggest that cationic liposomes, particularly DC-Chol/DOPE liposomes, will be useful as delivery agents for gene transfer to CNS cells in vitro and possibly in vivo.

Introduction

Gene therapy is an emerging technology for the treatment and biochemical elucidation of both genetic and acquired diseases. Rapid advances in detecting gene mutations responsible for disease and in understanding the molecular pathways involved have stimulated gene therapy research. Gene therapy entails the introduction of genetic material into a cell for the purpose of correcting a pathophysiological dysfunction.1 The use of genes in the role of a drug has many advantages over conventional drug treatment, including an increased specificity of treatment and the potential to offer a cure through precise replacement or correction of a malfunction. The majority of work to date has focused on targeting cells outside the central nervous system (CNS). This is due in part to the inaccessibility of the CNS caused by the blood–brain barrier as well as our limited understanding of the brain. However, the complexity of CNS disorders suggests gene therapy may be a valuable therapeutic alternative. In line with this, the first few gene therapy applications aimed at the CNS have recently been reported.2,3

While reasonable success has been achieved in moving to clinical trials with gene therapy for systemic diseases, gene therapy for the CNS has been hampered by the lack of efficient means for transducing postmitotic neurones. Naked DNA has been successfully used for gene transfer in limited tissues outside the CNS, however, its efficiency is rather low in the CNS. Most studies have utilised viral vectors for gene delivery. This is largely due to their enhanced efficiency over nonviral-based methods and the fact that they can infect neurones.4,5 Unfortunately, viral vectors are plagued by problems of immunity and cytotoxicity.6,7,8 In addition, the complex nature of producing viral vectors precludes them from widespread use in gene therapy. Nonviral vectors are now emerging as a popular method of cellular transduction. The most promising advances in nonviral gene transfer have been in the production of synthetic cationic liposomes formulated with cationic lipids (cytofectins) able to transfect cells. Such cationic liposomes are relatively easy to use, have a broad applicability and lack cytotoxicity.

At least 20 cationic liposome formulations have been identified which are successful in mediating transfection. However, few of these complexes have been examined for their ability to transduce cells efficiently within the CNS.9,10,11,12 Cationic liposomes act via electrostatic interactions with negatively charged DNA and subsequently with cellular membranes where they are taken across the cell membrane by a process of slow endocytosis.9,13,14 They are frequently formulated with the neutral lipid dioleoyl-L-α-phosphatidylethanolamine (DOPE) which is extremely efficient at endosomal buffering and disruption.11,15 From the perinuclear space transfected genetic material is released from the liposome complex, transported to the nucleus and expressed. To date, only liposomes formulated from N-[1-(2,3-dioleyloxy)propyl]-N,N,N trimethyl ammonium chloride (DOTMA) and DOPE, have been shown to successfully mediate transfection in the CNS.16,17,18,19 In order to be useful for gene therapy cytofectins capable of transfecting CNS cells with high efficiency are needed.

We have previously shown that cationic liposomes formulated from the cytofectin, 3β-(N-(N′,N′-dimethy- laminoethane)carbamoyl) cholesterol (DC-Chol) and DOPE are efficient at transfecting the neuronally derived ND7 cell line.20 DC-Chol has been used successfully outside the CNS in a variety of tissues and has recently undergone clinical trials for gene therapy treatments of cystic fibrosis.21,22 In addition, DC-Chol liposomes have been shown not to exhibit cytotoxic side-effects.23,24 For these reasons we wished to test further the applicability of the DC-Chol cytofectin for gene delivery to the CNS.

Owing to the uncertainties in designing novel liposome formulations each new liposome must be assessed for transfection capabilities individually in any given biological system. For this reason we have established a primary slice culture assay for assessing the optimal parameters of liposome-mediated transfection. This system is based on that of Stoppini et al25 and involves maintaining slices of CNS tissue in culture for prolonged periods. These slices have been shown to maintain the local biochemical, physiological and structural integrity of CNS regions in culture.25,26 For these reasons we predict these cultures will be a good model for testing novel cytofectins, which will work well in vivo. We chose to target the ventral mesencephalon since this region of the brain is implicated in the aetiology of neurodegenerative diseases such as Parkinson’s disease27 and has previously been cultured using similar methods.28 Furthermore, animal models of Parkinson’s disease have been developed which may be utilised to test novel cytofectins in vivo.29 In this initial study we report that DC-Chol/DOPE can successfully transfect organotypic brain slices of ventral mesencephalon. It is hoped that this work will enable the assessment of novel cationic liposome formulae that are highly efficient for CNS gene transfer.

Results

We successfully transfected organotypic explants cultured from ventral mesencephalon using DC-Chol/DOPE liposomes. These transfections were carried out using optimised plasmid DNA to liposome ratios previously identified for a neuronally derived cell line.20 Cultures generated from both newborn rat pups (P0) and 5 days postnatal (P5) were successfully transfected (Figures 1 and 2). No X-gal-positive cells were seen in cultures transfected with either plasmid or liposome complex alone (Figure 1). Although this was our first series of experiments with these cultures several important features of DC-Chol/DOPE-mediated transfection of nervous tissue emerged. First, of all, transfected cells displayed a variety of cellular morphologies. Multiple large cells exhibiting polarised dendritic arbours were positive for X-gal histochemistry (Figure 2). Smaller cells exhibiting short highly branched dendrites were also X-gal positive. Positively stained cells were found throughout the explant and did not coincide with any particular cellular population (ie substantia nigra). The morphological diverse nature of these transfected cells is consistent with the notion that DC-Chol/DOPE-mediated transfection targets both neuronal and non-neuronal cells. Second, the plasmid DNA/liposome complexes were able to penetrate into the explant (Figure 3). By 7 days in culture the explants had thinned to between 150 and 180 μm as determined by sectioning the explants in 10 μm intervals. X-gal-positive cells were routinely identified beneath the surface of the explant under high power magnification (Figure 3). In addition, explants subjected to serial sectioning revealed X-gal-positive cells up to 150 μm from the explant surface (Figure 3). Occasionally, positive cells were also observed in the deepest part of the slice, presumably via the millicell membrane (400 μm pores). Rarely were positive cells seen between 150 μm and 170 μm from the surface. This suggests that DNA/ liposome complexes were able to penetrate up to 150 μm from the superficial surface of the slice. No difference in penetration or thickness was seen between explants cultured for 7 versus 14 days (not shown).

Figure 1
figure1

Histochemical detection of X-gal in organotypic explant cultures of ventral mesencephalon from a 5-day-old rat pup (P5). Organotypic cultures were maintained for 1 week before being transfected with 2 μg pCMVβ complexed to 6 μg DC-Chol/DOPE. Slices were exposed to transfection medium for 1–2 h and X-gal histochemistry was performed 48 h later. Control cultures exposed to either DNA alone (a) or liposome alone (b) showed no X-gal positive staining. In contrast, numerous X-gal-positive cells were seen in cultures exposed to DNA–liposome complexes (c, d). Scale bar = 250 μm.

Figure 2
figure2

Successful transfections were also seen in organotypic explant cultures from ventral mesencephalon of newborn rat pups (P0). Organotypic cultures were maintained for 1 week before being transfected with 2 μg pCMVβ complexed to 6 μg DC-Chol/DOPE. Slices were exposed to transfection medium for 1–2 h and X-gal histochemistry was performed 48 h later. A variety of cellular morphologies were detected in transfected cells. Some transfected cells displayed long polarised arbours (a, c) while others displayed shorter more numerous arbours (b, d). Scale bar = 75 μm.

Figure 3
figure3

Plasmid DNA:DC-Chol/DOPE complexes are able to penetrate the explant culture. (a–c). Photomicrographs (×10) of a transfected whole slice (P5) taken at three successive focal points from the superficial surface illustrate several X-gal-positive cells located at various depths in the slice. In the most superficial figure (a) a long axonal process is the only X-gal-positive element in focus (arrowheads). When a deeper focal point was examined (b) the positive axon went out of focus while its corresponding cell body (arrowhead) and another cell (arrow) came into focus indicating they resided deeper in the slice. Focusing still deeper into the slice (c) caused the two cell bodies to go out of focus while another, deeper, positive cell became clear (arrowhead). (d, e) A more definite indication of how deep the transfection complexes penetrated the slice was obtained by examining X-gal-positive cells in consecutive 10 μm sections cut through a transfected slice culture (P5). (d) Two positive cells (open arrows) are clearly identified in this photomicrograph (×25) taken from a section 130 μm from the surface of the culture. (e) A higher power (×40) photomicrograph shows two positive cells (open arrow) taken 150 μm from the culture surface. The sections in (d) and (e) were counterstained with neutral red. Scale bar corresponds to 200 μm (a–c), 75 μm (d) and 45 μm (e).

It is difficult to determine the efficiency of transfection in these slice cultures owing to the large number of cells present and the potential heterogeneity between slices. However, in an attempt to gain a sense of relative efficiency between conditions we compared the total number of positive cells in a series of transfected slices through the ventral midbrain (Table 1). Ten sequential slices through half of the ventral midbrain from three different animals were compared for each condition. The total number of positive cells in the 10 slices were counted and averaged for each animal and condition (Table 1). Three different ratios of plasmid DNA to liposome complex were compared. All comparisons were performed with the same batch of liposomes and plasmid DNA preparation. As Table 1 illustrates a ratio of 1/3 DNA/liposome (w/w) was almost three times better than 1/1 and 1.8 times better than 1/2 (w/w) ratios. Higher ratios of liposome to DNA were no better than a ratio of 1/3 (w/w). A ratio of 1/3 DNA/liposome (w/w) corresponds to a 1/1 molar ratio of pCMVβ nucleotide/DC-Chol cytofectin (see Materials and methods). A 1/1 molar ratio in turn corresponds to a negative/positive charge ratio of approximately 1.0 since a nucleotide of anionic DNA and a molecule of the cationic DC-Chol cytofectin each contains a single charge.

In order to confirm successful transfections we performed a RT-PCR analysis. We designed oligonucleotide primers for the RT-PCR that flanked an intronic region downstream of the hCMV promoter in pCMVβ. Since the intron is excised during transcription amplification of cDNA during the RT reaction would produce a shorter PCR fragment (350 bp) than amplification of plasmid DNA alone (450 bp) (Figure 4a). Isolated RNA was not digested with DNAase to remove contaminating DNA so that amplification from both transfected plasmid DNA and transcribed product could be simultaneously detected. RT-PCR confirmed that we were achieving successful transfections in ventral mesencephalic tissue from P5 rat pups (Figure 4b). Similar results were achieved using ventral mesencephalic slices from P0 rat pups (not shown). A PCR product of 350 bp was detected in slice cultures transfected with pCMVβ:DC-Chol/DOPE complexes and not DC-Chol/DOPE alone, pCMVβ alone, or water (Figure 4b). Three different ratios of DNA to liposome were examined and all three gave positive results. In contrast to the results obtained from cell counts and X-gal histochemistry (Table 1) the RT-PCR results indicated that the 1/1 DNA/liposome (w/w) ratio worked better than 1/2, 1/3 or 1/4 (Figure 4). The PCR conditions used seemed to be within the linear range of amplification for the lower PCR product since using fewer cycles (30) produced no product (not shown). However, the saturating levels of plasmid DNA may have affected amplification of the RT-PCR product. Therefore, no conclusions other than a confirmation of successful transfection can be drawn from the RT-PCR experiments. It is likely that amplification of the larger PCR product was saturated and therefore masked variations which may be occurring with different DNA/ lipid ratios.

Figure 4
figure4

A RT-PCR analysis confirmed successful transfection of ventral mesencephalic cultures. (a) The reporter plasmid pCMVβ was used in all transfections. The primers used for PCR amplification flanked an intron (thicker line) in the hCMV promoter of pCMVβ which is excised during transcription thus generating two different sized products; a 450 bp fragment generated from transfected plasmid DNA and a 350 bp fragment generated from transcribed β-galactosidase RNA. (b) Ventral mesencephalic slice cultures were exposed to liposome–DNA complexes for 1 h then maintained for 48 h before being processed for total RNA extraction. Transfections were performed with 2 μg pCMVβ complexed to various DC-Chol/DOPE ratios (w/w). Lanes: (1) kb ladder; 2, naive slice; 3, no DNA control; 4, pCMVβ alone; 5, 1:3 no RT; 6, 1:1; 7, 1:2; 8, 1:3; 9, 1:4; 10, 0:2. The naive slice was treated in exactly the same manner as the experimental slices only without DNA or liposome. The arrows indicate a larger band (→, 450 bp) and smaller band (, 350 bp) corresponding to transfected plasmid and transcribed RNA, respectively. The small amount of 450 bp product in the null DNA control (lane 10) probably represents contaminating plasmid. No 350 bp product was ever detected in slices not exposed to pCMVβ. (c) In order to ensure that RT was performed on relatively equal amounts of RNA, cyclophilin was PCR amplified and analysed by Southern blotting. Lanes: 1, no RT; 2, naive slice; 3, 1:1 ratio; 4, 1:2 ratio; 5, 1:3 ratio; 6, 1:4 ratio; 7, 0:2 ratio. Only 15 PCR amplification cycles were performed in order to ensure that amplification was occurring in a quantitative manner.

Discussion

We report here, successful gene delivery to cells within the cultured rat ventral mesencephalon using the cytofectin DC-Chol and the neutral lipid DOPE. Cells displaying a variety of cellular morphologies were transfected indicating a certain degree of nonspecificity. Studies are currently underway to characterise these cell types better. A 1/3 (w/w) ratio of DNA/liposome (1/1 nucleotide/ cytofectin mol ratio) was determined to be optimal for transfection. In addition, we found that the transfection complexes were able to penetrate deeply into the slice culture. Successful transduction of ventral mesencephalon by DC-Chol/DOPE liposomes was confirmed using RT-PCR.

Optimising DNA to liposome ratios is critical for successful transfections.9 We found optimal transfection efficiencies with a DNA/liposome ratio of 1/3 (w/w), similar to that previously found for the ND7 neuronal cell line.20 It is not clear why this ratio is optimal since the negative/positive charge ratio is 1.0 (ie neutral). Cationic liposomes transfect cells via an endosomal pathway.19 Positively charged cationic liposomes spontaneously interact with negatively charged DNA.9 These DNA/ liposome complexes aggregate on the cell surface, probably through ionic interactions.13,19 Once bound to the cell surface the DNA/liposome complex is endocytosed or internalised and is then found within endosomal cellular compartments. When DNA/liposome ratios of 1/1 or 1/2 (w/w) are used, the complexes formed would have a net negative charge thereby reducing ionic interactions with the cell surface. Presumably this problem is overcome with charge neutral DNA complexes.

Direct comparison of these results with other studies is confounded by the use of different target cells, liposome formulations, and/or different reporter plasmid constructs. Slightly higher ratios of pCMVβ:DC-Chol/DOPE (1/5, w/w) are found to be optimal in epithelial cell lines when the complexes would be expected to have a net positive charge16 while negative/positive charge ratios of 1–0.8 worked best for DNA:DMRIE/DOPE complexes transfecting a kidney cell line (COS cells).9,13,14 In general variability between tissues dictates that each liposome formulation must be optimised for the target tissue. We have utilised organotypic explants of the CNS in order to develop a method of evaluating novel liposome formulations in a cellular environment as close as possible to in vivo conditions. However, care should be taken in attributing the findings here to in vivo conditions. Previous studies have shown that liposome formulations that work well in vitro do not always perform in vivo, and visa versa.30

Methods of gene transfer to the CNS in vivo are likely to involve a brief transient exposure of the target cells to the transfecting agent. For this reason our transfection protocol was limited to a single 1 h exposure of DNA/liposome complex to cultures. The effective exposure time of the cells to transfection complex is likely to be even lower since during this hour most of the transfection complex passed through the culture insert membrane. Even so, the transfection complex was able to penetrate through most of the slice. The transfection complex was able to penetrate approximately 80% of the slice from the superficial surface (ie 150 μm). This suggests that DNA:DC-Chol/DOPE complexes may be useful in vivo for transfecting CNS tissue. We are currently examining this possibility.

To our knowledge this is the first evidence of DC-Chol/DOPE-mediated transfection in a primary slice culture. In contrast to some commercially available cytofectins, DC-Chol has no major toxicity problems both in vitro and in vivo.24 It is because of its low levels of toxicity that DC-Chol has been utilised in phase I gene therapy trials in the United States of America.23 In this regard the present data are encouraging in that they show DC-Chol/DOPE liposomes are capable of transfecting neural tissue and in fact may even be successful in transfecting neuronal cells. This technology is poised to take advantage of the ongoing advances being made in our understanding of the molecular biology of neurological diseases. Further studies are now underway to characterise fully the cell types being targeted by DC-Chol/DOPE liposomes and to improve the overall transfection efficiencies. In addition, ways in which DC-Chol/DOPE can be targeted to specific neural tissues are also under investigation. It is hoped that these studies will eventually be applicable in whole animal models of neurological disease.

Materials and methods

Liposomes

DC-Chol/DOPE cationic liposomes were prepared by adding 6 μmol of DC-Chol and 4 μmol of DOPE (supplied at 10 mg/ml in CHCl3) to freshly distilled CH2Cl2 (5 ml) under nitrogen. Five millilitres of 20 mM Hepes (pH 7.8) was added to the mixture and this was sonicated for 3 min. The organic solvents were removed under reduced pressure and the resulting liposome suspension was then sonicated for a further 3 min. Liposome preparations were stored at 4°C.

Plasmid DNA

All transfections utilised the reporter plasmid pCMVβ (Clontech, Palo Alto, CA, USA) containing the full-length sequence for E. coli β-galactosidase downstream of the human cytomegalovirus immediate–early promoter/ enhancer (Clontech). Stocks of plasmid DNA were prepared from using standard molecular cloning techniques and purified over an anion exchange column (Qiagen, Dorking, UK).

Culturing solutions

Tissue culture media consisted of 47% minimal essential media (MEM), 24% Hank’s balanced salt solution (HBSS), and 24% heat inactivated horse serum, all purchased from Gibco BRL (Life Technologies, Paisley, UK). In addition, this basal medium was supplemented with 24 mM D(+)glucose, 0.94 mM CaCl2, 1.9 mM MgSO4, 28.3 mM Hepes (pH 7.2) from Sigma Chemical (Poole, UK) as well as 10 IU/ml penicillin–streptomycin (PenStrep; Gibco BRL). Serum-free medium was identical to culturing medium except that phosphate-buffered saline (PBS; Gibco BRL) replaced horse serum. Dissecting medium consisted of MEM supplemented with 10 IU/ml PenStrep.

Organotypic cultures

Ventral mesencephalic tissue was isolated from newborn (P0) and 5-day-old (P5) Sprague-Dawley rats. Rats were killed by rapid decapitation and their brains removed into ice-cold dissection buffer. The brains were then hemisected, placed medial side down, and dorso-ventral cuts made rostral to the inferior colliculus and through the caudal hippocampus. Any remaining cortical tissue and meninges were removed with forceps. The isolated mesencephalic tissue was then placed medial surface down on a McIllwain Tissue Chopper (Mickle Laboratory Engineering, Gomshall, UK) and 400 μm sections were made in the coronal plane. The tissue was then immediately transferred to pre-warmed culture medium where the slices were carefully separated and transferred on to pre-warmed 30 mm Millicell-CM culture plate inserts (Millipore, Watford, UK) with 750 μl of culture medium placed below the insert membrane. Three to four slices were divided into aliquots for each insert. The inserts were placed in 35-mm culture dishes (Nunc, Life Technologies, Paisley, UK). All cultures were maintained in a humidified incubator at 37°C with 5% CO2 and the culture medium was changed two to three times weekly by removing 500 μl and replacing it with fresh medium.

Transfection protocol

All organotypic cultures were maintained for 7–14 days before being transfected. The day before transfection the slice medium was replaced with serum-free medium. The transfection protocol was as follows: lipid complexes and reporter plasmid were divided into aliquots in separate vials and diluted with up to 200 μl serum-free medium. The diluted DNA was then slowly added to the diluted liposome complex, mixed gently and allowed to stand for 20–60 min. Immediately before being applied to the cultures DNA/liposome mixtures were brought to 1 ml with serum-free medium. DNA/liposome complexes were applied to the culture above the culture insert membrane and maintained at 37°C in a humidified incubator with 5% CO2 for 1 h. Following transfection, slice cultures were returned to fresh culture media (750 μl, below membrane).

X-gal histochemistry

Forty to 48 h following transfection slices were processed for histochemical staining for β-galactosidase activity. Following two brief washes in PBS, slices were fixed in 4% paraformaldehyde for 1 h, washed twice again in PBS and transferred to X-gal staining solution containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6. 6H2O, 1 mM MgCl2, 0.02% sodium deoxycholate, 0.02% NP-40, and 750 μg/ml X-gal in PBS. Slices were incubated in X-gal staining solution overnight at room temperature.

Frozen sectioning

Following X-gal histochemistry cultures were transferred to fresh 4% paraformaldehyde (para) and fixed overnight at 4°C. Cultures were then cryoprotected in 20% sucrose/4% para overnight (4°C) and either frozen for later sectioning or processed directly. Consecutive 10 μm frozen sections were cut in the coronal plane using a rotary freezing microtome, mounted on to polylysine-coated microscope slides (BDH Laboratory Supplies, Lutterworth, UK), and counterstained with neutral red. Subsequent photomicrography was performed using a Zeiss Axiophot2 microscope (Carl Zeiss, Welwyn Garden City, UK).

RT-PCR

Total RNA was isolated from slice cultures 48 h following transfection with DNA/liposome complexes according to the method of Chomczynski and Sacchi31 with slight modifications. Briefly, tissue was homogenised in 700 μl of solution D (4 M guanidium isothiocyanate; 20 mM sodium citrate (pH 5.2) 0.1% (v/v) β-mercaptoethanol) containing 0.033% (v/v) Antifoam A (Sigma) by repetitive pipetting. RNA was then purified by one round of water saturated phenol/CHCl3/IAA (25:24:1) extraction followed by isopropanol precipitation with 2 M NaOAc (pH 4.0). Total RNA was quantified by measuring absorbance at 260 nm.

Following isolation 2.5 μg total RNA was reverse transcribed (RT) with MMLV reverse transcriptase (20 units; Promega, Southampton, UK) and random hexamers (6.6 μg, Pharmacia Biotech, St Albans, UK) in 20 μl reaction buffer (50 mM Tris HCl (pH 8.3), 0.75 mM KCl, 0.3 mM MgCl2, 10 mM DTT) containing 10 units RNasin, (Promega) and 1 mM dNTPs. All reactions proceeded for 2 h at 37°C. Subsequently, 1 μl of RT reaction was processed for PCR amplification of E. coli β-galactosidase cDNA using the specific primers PCMVBGALFOR (5′-TTGACCTCCATAGAAGACACCG) and PCMVBGALREV (5′-CTGCAAGGCGATTAAGTTGGG). These primers were designed to amplify a region of pCMVβ which includes an intron immediately downstream of the cytomegalovirus promoter/enhancer as well as approximately 350 bp of 5′ coding region for E. coli β-galactosidase. The PCR conditions were 10 mM Tris.HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.1% (v/v) Triton X-100, 400 μM dNTP, 20 pmole each primer, and 2 units Dynazyme DNA polymerase (Flowgen Ltd., Lichfield, UK) in a final volume of 50 μl. Following an initial denaturation for 5 min at 95°C, the PCR reactions were subjected to 35 cycles of 94°C for 30 s, 57°C for 30 s and 72°C for 1 min, followed by 10 min at 72°C. The resultant PCR products were separated by electrophoresis on a 5% polyacrylamide gel and visualised with ethidium bromide.

As a control for the amount of total RNA used per RT reaction, PCR amplification of cyclophilin cDNA was carried out simultaneously. The cyclophilin primers were cycA 5′-TTGGGTCGCGTCTGCTTCGA and cycB 5′-GCCAGGACCTGTATGCTTCA. Cyclophilin PCR products were analysed by Southern blot rather than PAGE. As a result cyclophilin PCR reactions were carried out for 15 cycles rather than 35 in order to maintain a linear relationship between the amount of amplified DNA and the amount of initial RNA. Following PCR amplification, the DNA was separated by agarose gel electrophoresis and blotted on to Hybond-N transfer membrane (Amersham Life Sciences, Amersham, UK). Membranes were subsequently probed with a 32P end-labelled primer and hybridisation detected using film autoradiography.

Acknowledgements

This work was supported by grants from the MRC (G9426878N), The Middlesex Hospital Special Trustees (G.60), and the Motor Neurone Disease Association (Gurling/Apr 97/053).

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Correspondence to H M D Gurling.

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Murray, K., McQuillin, A., Stewart, L. et al. Cationic liposome-mediated DNA transfection in organotypic explant cultures of the ventral mesencephalon. Gene Ther 6, 190–197 (1999). https://doi.org/10.1038/sj.gt.3300743

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Keywords

  • gene therapy
  • β-galactosidase
  • DC-Chol
  • DOPE
  • primary culture

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