Establishment of an Atlantic salmon kidney cell line with an inducible gene expression system

Journal of Biotechnology 154 (2011) 209–211

Contents lists available at ScienceDirect

Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

Short communication

Establishment of an Atlantic salmon kidney cell line with an inducible gene expression system Bertrand Collet ∗ , Katherine Lester Marine Scotland, Marine Laboratory, 375 Victoria Road, Aberdeen AB11 9DB, UK

a r t i c l e

i n f o

Article history: Received 17 March 2011 Received in revised form 11 May 2011 Accepted 19 May 2011 Available online 26 May 2011 Keywords: Tet-off ASK Atlantic salmon recombinant Stable transfection

a b s t r a c t A stable recombinant Atlantic Salmon Kidney cell line ASK for use as an inducible expression system was isolated, cloned and characterised. The cells were transfected with the pTet-Off plasmid from the Tet On/Off Clontech system, carrying a G418 resistance gene. Several G418-resistant clones were sub-cultured and characterised by qPCR and by transient transfection. The level of expression of transcriptional activator (tTA) was measured by qPCR in a number of isolated clones. Transient transfection with a pTRE2-hyg-LUC plasmid was used to evaluate the inducibility of these clones. Two clones were chosen for their compromise between cell growth and inducibility. This genetically engineered cell line is a valuable tool for the fish research community especially in research areas investigating the biological function of viral proteins. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

Expression of a foreign gene in animal cells can be achieved by transfection with a plasmid containing a eukaryotic expressing cassette. In transfected cells, the plasmid if located in the nucleus directs the production of messengers. In some rare occurrences, random integration of the plasmid into the cell genome occurs. The integration of a positive selection marker such as G418 into the backbone of the expression construct allows for selection of stable recombinant cells to be isolated and propagated. The slow growth of fish cells makes the isolation of a recombinant cell line a long procedure but it gives access to an unlimited source of experimental material. In addition, unlike in transient transfection experiments, the expression of the transgene does not rely on high transfection efficiency, commonly very low in fish cells and showing large experiment to experiment variations. Constitutive promoter such as the CMV or SV40 early promoter direct high level of expression in cells from many species including fish. However, the over expression of certain viral or DNA-binding proteins is lethal to the cells and leads to their premature death. In addition, to investigate the function of a gene of interest by over-expression it is important to compare it to a control cell line over-expressing an irrelevant protein. This later remains imperfect as in most cases transgenes are integrated at different location in the genome resulting in different expression levels or different metabolism. In this respect, inducible eukary-

∗ Corresponding author. Tel.: +44 0 1224 425512; fax: +44 0 1224 295511. E-mail address: [email protected] (B. Collet).

otic expression systems are more advantageous. In the Tet-off tetracycline-inducible system, an artificial transcriptional activator tTA, encoded by the pTet-Off regulator plasmid consists of a 37-kDa fusion of amino acids 1–207 of the Tet repressor protein (TetR) and the C-terminal 127 amino acids of the Herpes simplex virus VP16 activation domain (AD; Triezenberg et al., 1988). The VP16 domain converts the TetR from a transcriptional repressor to a transcriptional activator, and the resulting hybrid protein is known as the tetracycline-controlled transactivator (tTA). In absence of tetracycline (Tc) or its derivative doxycycline (DOX), tTA binds the tetracycline-response element (TRE) in the response plasmid and activates expression of the gene of interest cloned into it. Expression level is therefore tightly controlled by the doses of Tc or Dox present in the medium. We report here the isolation, production and characterisation of a tet-off recombinant Atlantic salmon Salmo salar cell line, ASK-TOF3. The Atlantic salmon (S. salar) kidney cell line ASK (ATCC CRL2747 Devold et al., 2000) was grown as monolayer in complete culture medium: Leibovitz L-15 (Invitrogen) supplemented with 10% foetal calf serum (FCS, Invitrogen). The stable cell line was selected and maintained in ASK culture medium supplemented with 500 ␮g ml−1 G418 (Sigma) with or without DOX (Sigma). The initial cell line expressing the tTA regulator was obtained by transfecting ASK cells with the pTet-Off plasmid propagated into Escherichia coli (TOP10 competent cells, Invitrogen) purified using Endofree Maxiprep kit (Qiagen) according to the manufacturer’s instructions. After transfection, the cells were seeded at

0168-1656/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.05.010

B. Collet, K. Lester / Journal of Biotechnology 154 (2011) 209–211 40 35 30

Ct

25

X 18,283

X 14,032

20 15 10 5 0 ASK TOF 3 - RT

ASK TOF 3 + RT

ASK TOF 4 - RT

ASK TOF 4 + RT

Fig. 1. Level of expression of tTA in two ASK-TOF neomycin resistant clones. The level was corrected to ELF measured in the cDNA sample. The “−RT” control is a measure of the carry-over of genomic/plasmid DNA in RNA preparation. The Y-axis scale is logarithmic and the fold-increase in indicated over the “+RT” bar.

revived several months later, passaged 5 times until passage 20 and tested again by transient transfection with pTRE2hygluc as described above. After 46 h incubation at 22 ◦ C with doses of DOX of 0.5, 1, 10, 50 and 100 ␮g ml−1 the luciferase activity was significantly reduced (p < 0.01) and the percentage of

A 600

100%

500

400

RLU

very low density in culture medium containing G418. The cells were detached by Trypsin–EDTA (Invitrogen) action and pelleted by centrifugation at 5000 × g for 5 min and washed once with culture medium and twice with PBS. The pellet was drained and re-suspended in the cell resuspension solution R (Invitrogen Neon system, transfection kit) at a density of 1.5 × 107 cell ml−1 . The pTet-Off plasmid was added to half of the cell suspension at a concentration of 0.2 ␮g ␮l−1 and the other half were left without any DNA as a mock-transfected control. Two transfections were carried out in a multiporator MP100 using a 10 ␮l transfection kit (Invitrogen Neon system) set to a single 20 ms pulse at 1700 V, according to prior optimisation trials for this cell line (data not shown). The two sets of transfected cells were added to 5 ml of culture medium containing 500 ␮g ml−1 G418 in a 175 cm2 flask. After approximately 10 weeks, all the cells in the control flask were dead and in the Tet-off flask G418-resistant clones were approximately 1 cm in diameter. Four clones were collected and sub-cultured separately in 12.5 cm2 flasks. After 2 passages, one flask was used for qPCR characterisation. The total RNA was purified using the RNA/DNA/Protein purification kit (Qiagen) according to manufacturer’s instructions. The RNA was eluted in 50 ␮l RNase-free dH2 O. RNA was reverse transcribed to cDNA using the TaqMan® Reverse Transcription Reagent kit (ABI) with oligo-d(T)16 as follows: 9.625 ␮l of total RNA (approx. 0.5 ␮g) and 1.25 ␮l 50 ␮M oligo-d(T)16 were mixed and heated to 70 ◦ C for 10 min and chilled on ice. The final volume was adjusted to 25 ␮l by adding Master mix comprised of the following: 1× RT buffer (25 mM Tris–HCl pH 8.3, 37.5 mM KCl, 5.5 mM MgCl2 ), 0.5 mM each dNTP, 0.4 U RNase inhibitor and 1.25 U multiscribe reverse transcriptase or water for the mock RT control. Reactions were incubated at 48 ◦ C for 90 min, heat inactivated at 95 ◦ C for 5 min and stored at −80 ◦ C until use. Real-time PCR assays were performed on an ABI 7000 Sequence Detection System (Applied Biosystems). One microliter cDNA was added to the following mix contained in individual wells of a 96-well optical plate (Applied Biosystems): 10 ␮l of TaqMan® 2× PCR mix with UNG (Applied Biosystems), 8 ␮l of dH2 O and 1 ␮l of a 20× mix containing the forward primer (18 ␮M, 5 -CGCCCAGAAGCTAGGTGTAGA-3), reverse primer (18 ␮M, 5 -GGCTAAGGCGTCGAGCAA-3 ) and probe (5 ␮M, 5 6FAM-ACATTGTATTGGCATGTAAAAAATAAGCGGGC-MGB-3 ). The standard cycling conditions were 50 ◦ C for 2 min, 95 ◦ C for 10 min followed by 50 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min. The fluorescence output for each cycle was measured and recorded upon the completion of the entire run. Two clones showing a good cell division rate were sampled for measurement of the tTA gene expression level by qPCR. The level of expression of tTA was over 10,000 times the level found in the “−RT” controls (Fig. 1) showing a high level of transcription of the tTA expression cassette. To verify the inducibility of the ASK-TOF3 and 4 clones, the cells were transfected with the pTRE2-Hyg-Luc plasmid reporter (Clontech) using the same procedure described above, seeded in a 12 well plate and cultured with 0, 2, 20 or 40 ␮g ml−1 DOX for 48 h. The luciferase activity was measured by draining the cell culture medium from the plate, adding 75 ␮l of SteadyGlo luciferase substrate per well (Promega) cells and immediately measuring the light emission over 10 s using a Victor3 luminometer (Perkin Elmer). Luminescence intensity was recorded as Relative Light Unit (RLU). The addition of DOX, even at doses as low as 2 ␮g ml−1 resulted in a reduction of more than 95% of the luciferase activity in the clone ASK-TOF3 transiently transfected with pTRE2hyg-LUC (Fig. 2A). Only a 82% reduction was seen in the clone ASK-TOF4 (Fig. 2B). ASK-TOF3 cells showed the best compromise between growth rate and DOX inducibility. A passage 15 the cells were stored in liquid nitrogen in culture medium supplemented with 10% DMSO (Sigma). They were

300

200

100 4.3% 0

0

2

3.2%

2.0%

20

40

-1

DOX (µg.ml µ )

B

18 100% 16 14 12

RLU

210

10 8 6 4

18.3%

14.2%

17.6%

20

40

2 0

0

2 -1

µ DOX (µg.ml ) Fig. 2. Relative Light Units (RLU) in ASK-TOF clones transiently transfected with the pTRE2hyg-LUC plasmid and incubated without or with 2, 20 or 40 ␮g ml−1 Doxyclyclin (DOX) for 48 h. A. Clone ASK-TOF3; B. Clone ASK-TOF4. Data represent average RLU (N = 3) + SE. The % of the no DOX values is also indicated.

B. Collet, K. Lester / Journal of Biotechnology 154 (2011) 209–211

reduction was 88.2, 90.5, 92.1, 91.3, and 90.8%, respectively. This demonstrates the stability of the DOX inducibility in this cell line. This is the first report on the production of a tetracycline inducible expression system in an Atlantic salmon S. salar cell line. ˜ A similar system was generated previously in carp cells (Munoz et al., 2005; Rocha et al., 2004). Single recombinant cell lines producing luciferase had been established in the past as a tool to study specific sequences, to monitor toxicity of samples to fish cells (Richter et al., 1997) or to study signalling mechanisms but the transgene was always a reporter gene luciferase linked to a regulatory sequence or encoding an endogenous gene (Collet et al., 2004; Collet and Secombes, 2005; Martin et al., 2008). Previous attempts to force fish cell to produce ISAV proteins using plasmids with strong promoters such as CMV failed to produce any recombinant cells probably because of the toxicity of the viral proteins. Only transient transfection experiments were carried out (McBeath et al., 2006). This system is valuable to express potentially harmful proteins and monitor their effect in Atlantic salmon.

211

References Collet, B., Secombes, C.J., 2005. Construction and analysis of a secreting expression vector for fish cells. Vaccine 27, 1534–1539. Collet, B., Boudinot, P., Benmansour, A., Secombes, C.J., 2004. A luciferase reporter system based on Mx1 promoter to study interferon pathways in rainbow trout. Dev. Comp. Immunol. 28, 793–801. Devold, M., Krossay, B., Aspehaug, V., Nylund, A., 2000. Use of RT-PCR for diagnosis of infectious salmon anaemia virus (ISAV) in carrier sea trout Salmo trutta after experimental infection. Dis. Aquat. Org. 40, 9–18. McBeath, A.J.A., Collet, B., Paley, R., Duraffour, S., Aspehaug, V., Biering, E., Secombes, C.J., Snow, M., 2006. Identification of an interferon antagonist protein encoded by segment 7 of infectious salmon anaemia virus. Virus Res. 115, 176–184. Martin, S.A.M., Collet, B., MacKenzie, S., Evensen, O., Secombes, C.J., 2008. Genomic tools for examining immune gene function in salmonid fish. Rev. Fish. Sci. 16, 110–116. ˜ I., Carrillo, M., Zanuy, S., Gómez, A., 2005. Regulation of exogenous gene Munoz, expression in fish cells: an evaluation of different versions of the tetracyclineregulated system. Gene 363, 173–182. Richter, C.A., Tieber, V.L., Denison, M.S., Giesy, J.P., 1997. An in vitro rainbow trout cell bioassay for aryl hydrocarbon receptor-mediated toxins. Environ. Toxicol. Chem. 16, 543–550. Rocha, A., Ruiz, S., Estepa, A., Joll, J.M., 2004. Application of inducible and targeted gene strategies to produce transgenic fish: a review. Mar. Biotechnol. 6, 118–127. Triezenberg, S.J., Kingsbury, R.C., McKnight, S.L., 1988. Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev. 2, 718–729.