Stable transformation and long-term maintenance of transgenic Taxus cell suspension cultures
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- Ketchum, R.E.B., Wherland, L. & Croteau, R.B. Plant Cell Rep (2007) 26: 1025. doi:10.1007/s00299-007-0323-x
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A cell line of Taxus cuspidata has been transformed with wild-type Agrobacterium rhizogenes ATCC strain 15834 containing binary vector pCAMBIA1301 and, separately, with A. tumefaciens strain EHA105 containing binary vector pCAMBIA1305.2. Additionally, a cell line of T. chinensis has been transformed with wild-type A. rhizogenes ATCC strain 25818 containing binary vector pCAMBIA1301. The two transgenic T. cuspidata cell lines have been maintained in culture for more than 20 months, and the transgenic T. chinensis cell line for more than 9 months, with no loss of reporter gene expression or antibiotic resistance. The introduced genes had no discernable effect on growth or Taxol production in the transgenic cell lines when compared to the parent control. The methods for transforming non-embryogenic Taxus suspension cultures are described.
KeywordsTaxusTaxolPaclitaxelAgrobacteriumTransformationCell suspension culture
Cauliflower mosaic virus 35S promoter
High performance liquid chromatography coupled mass spectroscopy
Taxol (generic name paclitaxel) is arguably the most important anticancer agent to have been developed in the last 20 years, and the importance of this drug in the treatment of various types of cancers has been well documented (Long 1994; Holmes 1996). More recently, paclitaxel has shown promising results in drug-eluting coronary artery stents used in the treatment of diseased vasculature (Gruchalla and Nawarskas 2006). While the original source of Taxol, the bark of the Pacific Yew (Taxus brevifolia), limited its availability, recent means of production based upon extraction of the drug or its immediate precursors from cell culture or leaf tissue have helped to address the supply problem (see reviews by Patel 1998; Tabata 2004). However, because total synthesis of Taxol is not cost effective, isolation of the drug and its semisynthetic precursor 10-deacetylbaccatin III continue to rely on biological sources. The low abundance of Taxol and 10-deacetylbaccatin III in cell cultures and leaf tissue, and the complex purification procedures required, are responsible for the relatively high cost of the drug. Increasing the yield of Taxol and its precursors that can be obtained from natural sources would result in a more reliable supply of the drug and help to reduce the cost of this vitally important medicine.
Two groups have published preliminary reports on the transformation of Taxus (Han et al. 1994; Kim et al. 2000), although neither study provided unambiguous evidence of stable transformation, nor was the crucial requirement for long-term sustainability established. Here, we describe the successful transformation of a T. cuspidata and a T. chinensis cell line with either A. rhizogenes or A. tumefaciens containing one of two binary plasmids with an intron-containing β-glucuronidase gene, and report the subsequent maintenance and Taxol productivity over 14 months of stably transformed Taxus cell suspension cultures.
Materials and methods
Chemicals and reagents
Plant tissue culture medium, plant growth regulators, and amino acids were obtained from Caisson Laboratories (Logan, UT, USA). X-gluc (5-bromo-4-chloro-3-indolyl-β-d-glucuronide, sodium salt) and antibiotics were from Research Products International Corporation (Mt. Prospect, IL, USA) and Gold Biotechnology (St. Louis, MO, USA). All other chemicals and reagents were from Sigma-Aldrich (St. Louis, MO, USA).
Plant cell cultures
Cell suspension cultures of T. cuspidata, cell line P00E, were established from callus cultures initiated from embryos excised from seeds collected in the Finch Arboretum (Spokane, WA, USA) in September 2000. Plant cell cultures of T. chinensis were similarly established from excised embryos from seeds purchased from Sandeman Seeds (Lalongue, France). All Taxus cell cultures were initiated, maintained, and screened for Taxol production as previously described (Ketchum and Gibson 1996; Hezari et al. 1997; Ketchum et al. 1999, 2003). The TM19 medium used for culture of Taxus cells was modified from our previously described TM10 medium (Ketchum et al. 1995) by replacing the carbohydrates with 2% sucrose, replacing the vitamins with twice the concentration of Gamborg’s B5 vitamins (Gamborg et al. 1968), omitting the abscissic acid, and replacing the agar with 0.4% gellan gum. For liquid cultures, freshly prepared glutamine/antioxidant solution was added immediately prior to addition of cells as described previously (Ketchum and Gibson 1996). Transgenic cell suspensions were grown in TM19 containing 2.5 mg l−1 hygromycin.
Estimates of cell density in Taxus suspension cell cultures were made using a modification of the procedure described by Naill and Roberts (2004). Five milliliter aliquots of 7-day-old suspension cell cultures of Taxus were transferred to individual wells of a six-well plate (Falcon 353046, Becton Dickinson, Franklin Lakes, NJ, USA). Filter-sterilized Pectolyase Y-23 (Karlan Research, Cottonwood, AZ, USA) was added to each well to a final concentration of 0.5%, and cells were incubated in the dark at 25°C on a rotary shaker at 50 rpm. After 4 h of incubation, cell aggregates were further disrupted by gentle up and down pipetting in a Pasteur pipette. At least 20 fields of 1 mm2 were counted in a hemacytometer for each cell line.
pCAMBIA1305.2 (GenBank Acc. No. AF354046; Fig. 2) is derived from pCAMBIA1301, in which the E. coligusA gene has been replaced by the intron-containing GUSPlus gene, a β-glucuronidase gene from Staphylococcus sp. that is codon-optimized for in planta expression to give more rapid and thermally stable color development with 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-gluc). An additional feature of this vector is that a glycine-rich signaling peptide has been added to the GUSPlus gene to allow for extracellular excretion of the protein.
Binary vector pBI121 (Jefferson et al. 1987; Chen et al. 2003; GenBank Acc. No. AF485783) was obtained from Invitrogen (Carlsbad, CA, USA), and contains the nptII gene under the control of the nos promoter to confer resistance to kanamycin, and the gusA gene under transcriptional control of the CaMV35S promoter.
Several strains of both A. tumefaciens and A. rhizogenes were tested for efficient transformation of Taxus cell suspension cultures. A. rhizogenes ATCC strains 11325, 13333, 15834, and 25818 were obtained from the American Type Culture Collection (Manassas, VA, USA). A. tumefaciens strain EHA105 (Hood et al. 1993) was a gift of Prof. J. C. Carrington (Oregon State University). The Agrobacterium strains were made chemically competent and transformed with binary vectors using standard techniques (Sambrook et al. 1989).
A single colony of Agrobacterium selected on YM (Vincent 1970) agar plates containing 50 mg l−1 kanamycin was transferred and grown for 2 days at 28°C and 250 rpm in 2 ml liquid YM medium with kanamycin. Twenty-five milliliter of YM medium supplemented with 50 mg l−1 kanamycin and 50 μM acetosyringone was inoculated with 250 μl of this 2-day-old culture and grown overnight at 28°C and 250 rpm (OD600∼1.3). The bacterial medium was removed by centrifuging the entire culture at 3,000g for 15 min, and the resulting bacterial pellet was suspended in a 40 ml, 7-day-old Taxus culture in TM19 medium. Taxus cells and bacteria were incubated for 24 h at 125 rpm and 23°C. Taxus cells were washed by gently aspirating the medium with a 5 ml pipette, replacing with fresh TM19 medium containing 300 mg l−1 cefotaxime, and shaking for 15 min at 125 rpm and 23°C. This washing procedure was repeated three more times. Following the fourth wash, one or two grams of putatively transformed cells were plated on each of three to four 150 mm Petri dishes containing 25 ml of TM19 medium with 0.4% gellan gum and 300 mg l−1 cefotaxime. After 2 weeks of growth at 23°C, cells were transferred to selection medium consisting of TM19 with 300 mg l−1 cefotaxime and 2.5 mg l−1 hygromycin. Cells expressing the reporter gene could be stained for GUS activity after 2 weeks of growth on selection medium.
Following sufficient growth on agar medium, cell suspensions were reinitiated from callus and grown in TM19 with 300 mg l−1 cefotaxime and 2.5 mg l−1 hygromycin for 6 months, with subculturing every 2 weeks. After 6 months in suspension, cefotaxime was eliminated from the medium and the cells were grown in TM19 with 2.5 mg l−1 hygromycin.
β-Glucuronidase (GUS) activity was visualized by hydrolysis of X-gluc using a modification of the method of Castle and Morris (1994) in which the sodium salt dissolved in 0.2 M phosphate buffer, is substituted for the original cyclohexylammonium salt dissolved in N,N,-dimethyl formamide.
Southern blot analysis
Genomic DNA was isolated from 300 to 400 mg of each plant cell suspension culture using the DNeasy DNA extraction kit (Qiagen Inc., Valencia, CA, USA), according to the manufacturer’s instructions. Buffers and procedures for Southern blot analysis were according to Sambrook et al. (1989). Seven micrograms of genomic DNA from control and transgenic cultures were digested overnight with HindIII or XbaI (New England Biolabs, Cambridge, MA, USA) at 37°C; each enzyme cutting once within the T-DNA region of the plasmid. Digested DNA was electrophoresed on a 0.8% TAE-agarose gel in TAE buffer, for 16 h at 20 V. DNA was transferred to a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Pisscataway, NJ, USA) according to Sambrook et al. (1989) and fixed by UV cross-linking. A 500 bp fragment of the gusA gene was prepared by PCR amplification of pCAMBIA1301 with the following forward primer, 5′-GCAGCGTAATGCTCTACACCACGCCGAAC, and reverse primer, 5′-TGCCATGTTCATCTGCCCAGTCGAGC. This DNA fragment was labeled with β-[32P]dCTP (Ready-to-Go Labeling Beads, Amersham Pharmacia Biotech) and residual unincorporated nucleotides were removed using ProbeQuant G-50 Micro Columns (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Following overnight hybridization at 65°C, membranes were washed according to standard procedures (Sambrook et al. 1989) and Kodak X-Omat Blue XB-1 film was exposed to the hybridized membrane at −80°C for 14 days.
Taxoid extraction from cells and HPLC analyses
Minor modifications in sample preparation were made to our previously described method for routine analytical screening of cell suspensions (Ketchum et al. 1999). A 250 μl aliquot of cell-free suspension medium was placed in the lower portion of a Mini-Uniprep filter/autosampler vial (Whatman, Florham Park, NJ, USA). An equal volume of CH3CN was added to the sample, the vial was capped with the filter portion of the unit by pushing the upper assembly partially into the lower assembly, and the sample was vortexed thoroughly and filtered by completely depressing the upper portion of the assembly. HPLC-MS analysis of the taxoid content of cells and suspension medium was as previously described (Ketchum and Croteau 2006).
Estimates of transformation efficiency with non-embryogenic suspension cell cultures are particularly challenging in comparison to the transformation of explant sources used for the eventual regeneration of transgenic tissue. Unlike experiments with typical explants, such as leaves or embryos, a non-embryogenic suspension is composed of millions of undifferentiated cells and cell aggregates, each cell being the potential explant. In the three successful experiments described in this report, an average of 24 transformed calli was obtained per 2 g fresh weight of plated cells. Estimates of cell density in 7-day-old cultures of P00E are 2.7 × 105 cells ml−1 (1.9 × 106 cells g−1) and 7.3 × 104 cells ml−1 (5.1 × 105 cells g−1) for cell line H04C. These cell density estimates are consistent with values of 0.5 to 3 × 106 cells g−1 determined by Naill and Roberts (2004) for our previously described Taxus cell lines C93AD, P991, P93AF (Ketchum and Gibson 1996) and P093X (Ketchum et al. 1999). Thus, transformation efficiency is on the order of 0.0006% for P00E and 0.002% for H04C. Transient expression was much more common, and could usually be detected in 50% of the transformation experiments. However, obtaining a stable cell culture following the detection of transient expression proved exceptionally difficult. From the first experiment where actively growing transgenic callus was obtained, 113 transformation experiments were performed before another actively growing transgenic callus line was obtained. A more realistic estimate of transformation efficiency, therefore, may be that approximately 1% of experiments result in a stable, maintainable, transgenic cell line.
Literature reports of successful transformation of non-embryogenic suspension cultures in general, and of gymnosperms in particular, are limited (e.g. Nakamura and Ishikawa 2006; Tian et al. 2000). Because regeneration of Taxus is unnecessary for production of taxoids in culture, we decided to test wild-type strains of both A. rhizogenes and A. tumefaciens because there was no need to have a disarmed Agrobacterium strain for transmission of the vector. A. rhizogenes ATCC 15834 is an agropine-type strain (Petit et al. 1983) that has been used extensively for the development of hairy root cultures. A. tumefaciens EHA105 is a disarmed strain that is commonly used in the transformation of dicotyledonous species (Hood et al. 1993). Both these Agrobacterium strains were successfully used for transformation of Taxus cells.
Preliminary experiments demonstrated that Taxus cell cultures were resistant to concentrations of kanamycin typically used for selection with dicotyledonous species, e.g. 50 mg l−1 with tobacco cells (Fraley et al. 1983). The high proportion of non-transformed escapes that we obtained using kanamycin as a selection agent, and the ability of Agrobacterium cells containing pBI121 to exhibit β-glucuronidase activity, led us subsequently to test available pCAMBIA binary vectors with intron-containing GUS reporter genes and hygromycin resistance genes (Fig. 2). “Escape” colonies that were frequently encountered with kanamycin selection were eliminated by using hygromcyin at a concentration of 2.5 mg l−1, considerably less than the 10 mg l−1 concentration used by Kim et al. (2000). The intron in the gusA or GUSPlus gene eliminated the possibility that the bacteria were responsible for positive GUS assay results. Further confirmation of the T-DNA integration into the Taxus genome was demonstrated through Southern hybridization analysis (Fig. 7).
One of the challenges of using cell culture technology to produce useful amounts of Taxol from Taxus tissue cultures has been the establishment of healthy cultures. As has been well documented, Taxus plant cells are difficult to manipulate in vitro (Ketchum and Gibson 1996), and the additional stress imparted by transformation compounds the difficulty (Kim et al. 2000). T. cuspidata cell line P00E, although one of our lowest Taxol producing cell lines, grows robustly in culture and was used for many of our initial transformation experiments because it survived incubation with Agrobacterium with little noticeable effect, as opposed to the rapid reddening or browning that occurred in cultures of T. x media or T. canadensis.
Because these transgenic Taxus cells have been monitored continuously since these suspension cultures were established, we have detected no influence on Taxol production in the transgenic cultures due to the introduced genes (Fig. 8). Additionally, there were no detectable differences in the taxoid profile of the transgenic cultures compared to the untransformed control when cell and medium extracts were analyzed by HPLC-MS (data not shown). The fluctuation in Taxol productivity over time in both transgenic and non-transgenic Taxus cells is remarkable, in that the variability is independent of cell genotype and may indicate that environmental factors are responsible for this frequently observed occurrence in Taxus cell cultures (Ketchum and Gibson 1996). However, the equally plausible explanation that variability is genetically derived from the parent P00E cell line, but still unaffected by transformation, cannot be discounted.
If transgenic Taxus cell cultures are to be used for large-scale production of Taxol, then it is essential that these cultures grow as rapidly as the parent cells, and that they stably express the genes of interest for the extended time necessary for scale-up. For example, under our culture conditions, it takes 10 weeks to scale-up a 40 ml culture to produce sufficient biomass to inoculate a 125 l (final volume) culture. Our results indicate that it is possible to obtain and maintain rapidly dividing transgenic Taxus cell suspension cultures, and that the integrated genes do not necessarily affect growth or Taxol productivity when compared to the parent control.
Clearly, based on our “best” transformation efficiency of 1% of transformation experiments resulting in a stable transgenic Taxus cell line, transformation of Taxus is achievable although not yet routine. Experiments are underway to identify those factors critical for the successful and reliable transformation of Taxus. That one species of Taxus can be transformed with either a disarmed strain of A. tumefaciens or a wild-type strain of A. rhizogenes, and that a second can be transformed with an additional wild-type strain of A. rhizogenes, suggests that testing the many possible permutations of Taxus species and Agrobacterium strains may be one of the factors that are important for developing a more optimal transformation protocol.
The establishment of transgenic cell technology in Taxus is the first step toward metabolic engineering of the Taxol biosynthetic pathway, both to better understand the complex pathway and, ultimately, to improve drug production.
This investigation was supported by U.S. National Institutes of Health grant CA-55254, and by McIntire-Stennis Project 0967 from the Washington State University Agricultural Research Center. The authors wish to thank James Green and Carina Ng for technical assistance and Mark Wildung for insightful discussions.