A rapid and easy method to clean and concentrate adenoviruses for in vitro and in vivo applications.

INTRODUCTION

The use of Adenoviral vectors (Ad-vectors) encoding GFP (Green Fluorescent Protein) or various other marker proteins, for example RFP (Red Fluorescent Protein), is very useful for many studies in the central nervous system including tract-tracing studies (1-2), intraventricular injections (3) or ex vivo cell labelling for cell transplantation purposes. (4) However, the purification and concentration of adenoviruses is a very difficult procedure, because according to the classical cleaning and concentration steps ultracentrifugation with CsCl-gradients is needed. (5,6,7) Centrifugation is time consuming and expensive since ultracentrifuges are needed and toxicity due to CsCl-contamination cannot be excluded. (6)

Here we describe a rapid, non toxic and easily to perform method that can be practically applied in almost every laboratory for neuroscientific research with the appropriated security level for handling adenoviruses by the use of membrane chromatography. (8)

MATERIALS AND METHODS

Adenovirus propagation

All preparation dealing with adenoviruses has been performed in a biosafety level 2 facility equipped with a biological class 2 safety cabinet. Adenovirus type 5 (Adeno-X[TM] AcGFP1 Marker Virus) encoding the green fluorescent protein (GFP) under the immediate early promoter of human cytomegalovirus was obtained from Clontech Laboratories (Mountain View, Ca, USA). This virus lacks the E1 gene and, therefore it replicates only in E1 trans-complementing cells such as HEK293-cells. HEK293-cells were cultured and expanded in Petri dishes (9.2 cm diameter, NUNC, Hereford, UK) containing DMEM, 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 [micro]g/ml streptomycin (all media and supplements from Invitrogen, Karlsruhe, Germany) in a humidified incubator (95 % air/5% C[O.sub.2], at 37[degrees]C). 72h after adenoviral transduction HEK293-cells were scraped from the bottom of the Petri dishes and immediately, together with the supernatant frozen at -80[degrees]C, until further processing.

Adenovirus purification and concentration

Adenovirus purification and concentration by using the Vivapure[R] AdenoPack[TM] 100 kit was performed with the supplied material and according to the manufacturer's instructions (Vivascience AG, Hannover, Germany). In brief, for purification, cells and supernatant from three Petri dishes containing about 20 ml solution were frozen at -80[degrees]C and subsequently thawed three times at 25[degrees]C. Then the cell debris was centrifuged (Megafuge 1.0R, Heraeus, Dusseldorf, Germany) at 3500g for 15 min and 20 ml of the supernatant, containing the viruses, was incubated for 30 min at 37[degrees]C with 20 [micro]l Benzonase[R]. The solution was then transferred to a Vivaclear Maxi tube, centrifuged at 500 g for 15 min and the flow through was gently mixed with the loading buffer according to the manufacturer's protocol. After equilibration and wash through of the AdenoPACK Maxi spin column with washing buffer the column was then filled up with the loading buffer containing the viruses and centrifuged at 500 g for 5 min. The AdenoPACK Maxi spin column was then again rinsed two times with washing buffer followed by centrifugation at 500 g for 5 min. The membrane bounded adenoviruses were then, after incubation for 10 min in elution buffer, centrifuged at 500 g for 5 min and the adenovirus containing eluate was collected. To exchange the buffer and further concentrate the viral solution the eluate, about 1 ml, was transferred to a Vivaspin 20 concentrator tube, filled up with about 9 ml of sterile phosphate buffered saline (PBS, 0.1M, pH 7.4) and centrifuged at 800 g for 30 min. The amount of concentrated viruses in several experiments was about 230 [micro]l. The titer of the adenoviral solution was then determined by using an Adeno-X[TM] rapid titer kit (Clontech Laboratories, Mountain View, Ca, U.S.A.) and was then adjusted to 1 x [10.sup.7] ifu (infectious units) per [micro]l with sterile PBS.

Intraventricular Ad-vector injections

A total of 9 male C57BL/6-mice weighting about 28 g at beginning of the experiment were housed at 22 [+ or -] 2[degrees]C under a 12 h light/dark cycle with free access to food and water. All animal-related procedures were conducted in accordance with NIH and local ethical guidelines and have been approved by the animal experimentation committee of the University of Rostock.

Mice were deeply anaesthetized by an intraperitoneal injection of Ketamin (75 mg/kg) and Rompun (5.8 mg/kg) and mounted in a mouse adaptor (Stoelting Co) fixed in a rat stereotaxic apparatus (Kopf, Tujunga, USA). The skull was opened with a dental thrill and animals received the viral solutions (0.3 [micro]l, 1[micro]l or 3 [micro]l) containing about 1 x [10.sup.7] ifu per [micro]l by an injection into the right lateral ventricle via a glass capillary with an outer diameter of about 80 [micro]m mounted on to a 26 ga Hamilton syringe, coordinates referring to bregma: AP [+ or -]0, ML -0.8, V -1.8 (Dura). (9) After injection of the solutions (0.3 [micro]l per min) the syringe remained for further 3 min in the ventricle, was then slowly retracted and the skin was sutured.

[FIGURE 1 OMITTED]

Tissue processing

Three days after virus injection mice were injected with an overdose of pentobarbital-Na+ (60 mg/kg) and transcardially perfused with ice cold 0.9% sodium chloride (10 ml), followed by 50 ml of 3.7% PFA. Brains were immediately removed from the skull, postfixed overnight, and transferred into 0.1 M PBS containing 20% sucrose (overnight, 4[degrees]C). The cryoprotected brains were frozen in isopentane (-50[degrees]C) and stored at -80[degrees]C. The brain tissues were placed in a cryostat and 30[micro]m sections were cut, free-floating in PBS, mounted on to glass slides and finally embedded with a fluorescence mounting medium.

Cell transfection

Conditionally immortalized CSM14.1-cells were cultured under differentiation conditions as described earlier (10) in 3 cm diameter Petri dishes (Nunc) containing DMEM supplemented with 1% FCS, 100 U/ml penicillin, 100 [micro]g/ml streptomycin (all reagents from Invitrogen, Karlsruhe, Germany) in a humidified incubator (95% air, 5% C[O.sup.2], at 39[degrees]C). About 106 cells were transfected with 1 x [10.sup.7] ifu of the purified adenoviruses and GFP-expression was documented 72 h later.

Analysis and documentation of GFP-expression

Documentation of tissue slices was performed with a confocal laser scanning microscope (Nikon Eclipse E400 with the confocal system C1) or for cell cultures with an inverted microscope (Nikon Eclipse TS100F, Badhoevedorp, The Netherlands) equipped with a digital camera (Coolpix 4500, Nikon, Tokyo, Japan).

RESULTS

The injection of purified Ad-vectors into the lateral ventricles of adult mice leads to the transduction of ependymal cells aligning the lateral ventricles (Fig. 1A-F). Moreover, the differences depending on the amount of injected Ad-vectors (0.3 [micro]l, 1 [micro]l or 3 [micro]l) in number of transduced cells and their intensity of GFP-expression was obvious (Fig. 1A-F). By the hereby applied amount of Advectors we observed no toxic effects on the transduced cells. The use of Ad-vectors for the transduction of cultured CSM14.1-cells also lead to a strong and robust GFP-expression without any signs of neurotoxicity (Fig. 1G-I).

DISCUSSION

The use of membrane chromatography (8) has been proven to be a rapid and non toxic method to clean and concentrate Ad-vectors for in vivo and in vitro applications. In contrast to the time consuming ultracentrifugation with repeated CsCl-gradients (5,6,7) the presented method for cleaning and concentration of Ad-vectors was performed within 2-4 h with high yields. Moreover, the contamination of the viral solution with a toxic compound is not possible anymore because CsCl was omitted. (6) In comparison to commercially available Ad-vectors the presented cleaning and concentration procedure is very cheep and can be performed in laboratories by scientists that do not have access to ultracentrifuges.

ACKNOWLEDGMENTS

We acknowledge Nicole Ludke for her skilfull technical assistance. Our work was supported by the FORUN grant No 889011/2008 of the Medical Faculty, University of Rostock to S.J.-P.H.

REFERENCES

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Corresponding author: Stefan Jean-Pierre Haas, Institute of Anatomy, University of Rostock, Gertrudenstrasse 9, D-18055, Rostock, Germany, Tel: [phone], fax: [phone], ?-mail: [e-mail]

Steve Hildebrandt, Stefan Jean-Pierre Haas, Christian Andressen and Andreas Wree

Institute of Anatomy, Medical Faculty, University of Rostock, Rostock, Germany