Efficient transformation of mutant cells of Chlamydomonas reinhardtii by electroporation

Efficient transformation of mutant cells of Chlamydomonas reinhardtii by electroporation

Process Biochemistry 39 (2004) 1685–1691 Efficient transformation of mutant cells of Chlamydomonas reinhardtii by electroporation Vladimir G. Ladygin...

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Process Biochemistry 39 (2004) 1685–1691

Efficient transformation of mutant cells of Chlamydomonas reinhardtii by electroporation Vladimir G. Ladygin∗ Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, ul. Institutskaya 2, Moscow 142290, Russia Received 31 March 2003; accepted 19 July 2003

Abstract Cells of CW-15 mutant of Chlamydomonas reinhardtii without a cell wall were transformed by electroporation. The hpt gene of hygromycin phosphotransferase was used as a selective marker. Optimal conditions of transformation were observed in the middle of the logarithmic growth phase at the density of suspension 106 cells/ml, electric field intensity 1 kV/cm, and pulse duration 2 ms. Under these conditions up to 103 hygromycin-resistant clones of trasformants per 106 recipient cells were obtained that was 100 times higher than at the usage of wild-type cells. Exogenic DNA integrated into the genome of the nucleus C. reinhardtii was constantly inherited for more than 350 generations. The use of mutants without a cell wall and certain selective systems enable the efficiency of transformant yield to be doubled problems on unstable expression of geterologous genes to be investigated, and ways of obtaining super producers of foreign proteins using the alga C. reinhardtii investigated. © 2003 Elsevier Ltd. All rights reserved. Keywords: Chlamydomonas reinhardtii; Mutant CW-15 without a cell wall; Transformation; Gene hpt – hygromycin phosphotransferase; Electroporation

1. Introduction The eukaryotic unicellular green alga Chlamydomonas reinhardtii Dang, is a convenient model for use in studying biological processes, including chloroplast structural organization and functional activity, cell interactions, conjugation type, cell cycle, and photosynthesis. In addition, due to its ability to grow autotrophically on inexpensive mineral nutrient media, this alga is a potential superproducer of foreign proteins. Presently, such production is impossible because heterologous genes are not readily expressed in C. reinhardtii cells. In most studies on C. reinhardtii nuclear genomes transformed with foreign genes, the bacterial nptII gene has been employed as a selectable marker [1]. This choice is not the best since C. reinhardtii is known to exhibit a high frequency of spontaneous kanamycin-resistant mutations [1–3]. Moreover, the first study on the transformation of wild-type C. reinhardtii cells demonstrated a very low output of trans-

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formed cells [4]. To overcome both drawbacks of C. reinhardtii, the hpt gene was employed as a selectable marker with a CW-15 mutant devoid of cell walls to enhance DNA entry by electroporation. Genetic transformation of C. reinhardtii was reported as early as 1982 [4]; however, no significant progress was achieved in this direction until recently after the development of several new technologies for transforming cells of microorganisms [5,6] and plants and animals [7]. Thus, the biolistic technique (cell bombardment with DNA-coated tungsten microscopic particles) was used to transform C. reinhardtii chloroplasts [8]. The nuclear genome was effectively transformed by rigorously agitating a cell suspension with plasmid DNA and glass beads [9]. Both chloroplast and nuclear homologous genes of C. reinhardtii were used by these and other authors. Stable transformed plants were obtained by complementing the mutant chloroplast atpB [8,10], rbcL and psbA [11], and psaB [12] genes with wild-type sequences. This approach employs a homologous recombination between the mutant gene and the cloned wild-type sequence. Mutant chloroplast genes, which encode 16S rRNA and 23S rRNA and confer the corresponding resistances to specti-


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nomycin and streptomycin, and also to erythromycin, were used to select transformed clones resistant to antibiotics [10,11,13]. Nuclear DNA was transformed with the homologous nit1 [9,14], oee1 [2], rsp3 [9,15], and argl [16] genes. The use of heterologous genes for C. reinhardtii transformation meets several difficulties. Sustainable expression of the p-glucuronidase (uidA) gene from Escherichia coli fused with the 5 -untranslated region of the chloroplast petD gene from C. reinhardtii [17] and the aminoglycoside adeninetransferase (aadA) gene from E. coli was reported to confer C. reinhardtii resistance to spectinomycin and streptomycin [18]. However, the expression of heterologous genes integrated into the nuclear genome remains a complicated problem for future solution. The scarce data available on nuclear genome transformation by heterologous genes in C. reinhardtii are inconsistent and do not elucidate the cause of unstable expression. In the pioneering 1982 study on C. reinhardtii transformation, the yeast argininosuccinate lyase (arg4) gene was used to complement the ARG7 mutant [4], with the nptII gene from the bacterial transposon Tn5 as a selectable marker [1]. The problems that arise when these two selection systems are used have already been discussed [2,3]. Fifty two per cent of clones transformed with a plasmid carrying two selectable markers, nptII and nit1 were reported to express the nptII gene [3]. In the latter case, transformed cells were selected on a medium with nitrate as the sole source of nitrogen. Apparently, some of the constraints observed when C. reinhardtii cells are transformed with foreign genes are due to the Km selectable marker [3,13], particularly because spontaneous kanamycin-resistant mutations arise at a high frequency. In this study, we constructed and characterized the pCTVHyg plasmid and demonstrated that the CW-15 mutant cells of C. reinhardtii are feasibly and efficiently transformed by electroporation using the hpt gene from E. coli as a selectable marker.

2. Materials and methods 2.1. Microorganisms The heterothallic single-cell alga C. reinhardtii grows rapidly on mineral medium, has a reproductive cycle, large oval cells, 10–15 mm in diameter and a biochemical composition similar to the cells of higher plants. The alga has one chloroplast and can be used as a convenient model for biotechnological purposes, since it is easy to obtain mutants, transformants and great biomass increase. A suspension of CW-15 mutant cells of C. reinhardtii was used for transformation. In contrast to the wild-type cells (strain 137C mt+), mutant cells do not develop cell walls [19].

2.2. The nutrient medium and culturing conditions Algae were cultured in a liquid medium (a minimal Levine and Ebersold [20] medium modified in this laboratory) bubbled intensely with 5% CO2 under constant illumination (4 klx) at a temperature of 25 ◦ C [21]. To solidify the medium, it was supplemented with 1.5% Difco agar (Difco, USA). 2.3. Transformation CW-15 mutant cells were transformed by breaking biological membranes with a rectangular current pulse. To transform large algal cells (10–15 ␮m), a purpose made pulse generator (Kaunas, Lithuania) Medical Academy vas used. An algal culture grown to a density of 106 cells/ml was centrifuged at 1500 × g for 5 min and washed in the same medium. Cell numbers were counted in a Goryaev cytometer. The cell concentration was then increased 100-fold by recurrent centrifuging and resuspension in an electroporation buffer (10 mM Tris–HCl, pH 7.5, 10 mM CaCl2 , and 0.4 M mannitol). Plasmid DNA (5 ␮g) and salmon sperm DNA (10 ␮g) were added to the suspension (only salmon sperm DNA was added to the control suspension). Suspension aliquots 0.4 ml in volume from each treatment were transferred into an electroporation chamber (the distance between the electrodes was 1 cm) and treated with a 1 kV/cm pulse for 2 ms. Cells were kept on ice for 10 min and then incubated in light for 16–18 h. Next, cells were collected by centrifugation at 1500 × g for 5 min and resuspended in 1 ml of the medium. To plate cells on Petri dishes, 0.1 and 0.5 ml aliquots of suspension were melted into a soft agar medium; the latter comprised a mineral nutrient medium containing 10 ␮g/ml of hygromycin B (Calbiochem, USA) per ml and 0.6% Difco Bactoagar. Cells were incubated for 10–14 days in light at 25 ◦ C. 2.4. Total DNA isolation and Southern hybridization Total DNA was extracted from 20 ml of the culture grown to late logarithmic phase as previously described [22]. Hybridization was carried out as previously described [23] using nylon filters (Hiiyu–Kalur Experimental Laboratory, Estonia). Probes were prepared from a BamHI fragment of the pPCV730 plasmid (a gift from J. Shell, Max-Planck-Institut fiir Zuch-tungsforschung, Koln, Germany) comprising the coding region of the hpt gene and labeled by random priming with a Boehringer Mannheim (Austria) kit following the manufacturer’s instructions. 2.5. Assay of NPTII activity CW-15 mutant cells were suspended in 10 ml of a liquid mineral medium to a final density of 106 cell/ml, transformed with plasmid DNA, and incubated for 18 h in light. NPTII

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activity was assayed in a crude extract of C. reinhardtii cells as described previously [24].

3. Results


nptII coding sequence [26]. The sticky ends were repaired using the Klenow fragment. Cells of E. coli, strain HB101, were transformed with the ligase mixture, and bacterial clones with direct nptII orientation were selected. In this way, the pCTVNeo plasmid for C. reinhardtii transformation (Fig. 1) was obtained.

3.1. Plasmid engineering Using a heterologous gene, several integrative vectors were constructed. Bacterial genes of E. coli, hpt [25] and nptII from the Tn5 transposon [1,26], were chosen as selectable markers. The Chlamydomonas transforming vector pCTVHyg with the hpt selectable marker conferring HygR was constructed in the following way (Fig. 1). The initial pSV2Cat plasmid [27] was digested with HindIII and HpaI, and the 3.5-kb fragment with the promoter region and the SV40 viral polyadenylation site isolated and ligated with a 1.1-kb /BamHI-cut fragment from a pPCV730 plasmid comprising the coding sequence of the hpt gene [25]. Sticky ends were preliminarily filled in using the Klenow fragment. The ligase mixture was used to transform E. coli, strain HB101, and clones with direct hpt orientation were selected. All these steps were performed following published protocols [28]. The transforming vector pCTVNeo with the nptII selectable marker conferring kanamycin resistance was produced from the pSV232A-L plasmid [29] by restriction with HindIII and SmaI. A 6.6-kb fragment of the plasmid comprising the promoter of early genes and the polyadenylation site of the SV40 virus was ligated with a 1-kb BglII-BamHI fragment from the pMON129 plasmid [30] representing the

3.2. Transformation of CW-15 mutant cells of C. reinhardtii To transmit exogenous DNA to CW-15 mutant cells, an electroporation method of transformation was developed with the pCTVHyg plasmid. This plasmid comprises the coding region of the hpt gene under the promoter of early SV40 viral genes. To choose the optimum transformation conditions, a C. reinhardtii cell suspension was subjected to electric pulses of various duration and amplitude [31,32]. Transformation was most effective at an electric field voltage of 1 kV/cm and pulse duration of 2 ms (Fig. 2). Following 4–5 days of growth on the selective medium under constant illumination, the death of most hygromycinsensitive cells was observed microscopically. HygR colonies were visible after 8–10 days of cell growth. Transformation efficiency was about 103 HygR transformant clones per 106 recipient cells. The efficiency of transformation depended considerably on the phase of culture growth. Highest output was observed with culture in the mid-logarithmic growth phase at a suspension density of 106 cells/ml. At a suspension density exceeding 3 × 106 cells/ml, transformation was negligible (Fig. 3). 3.3. Analysis of transformed clones Following 8-month-long cultivation (clones were transferred to new plates every 14 days on nonselective medium), total DNA was isolated from four HygR clones, digested with PstI, fractioned in 0.8% agarose gel, blotted onto a nylon filter and hybridized with the BamHI-cut fragment of the pPCV730 plasmid comprising the coding hpt sequence. Hybridization of two such clones (Fig. 4, lanes 2

Fig. 1. The structure of initial plasmid pSV2Cat and derivative plasmids pCTVHyg and pCTVNeo linearized at the EcoRI site. CAT: chloramphenicol acetyltransferase; HPT: hygromycin phosphotransferase; NPTII: neomycin phosphotransferase; AmpR : resistance to ampicillin; PSV40 : promoter of the early SV40 viral genes; pA: SV40 viral polyadenylation site. Restriction sites: E, EcoRI; Ps, PstI; Pv, PvuII; H, HindIII; Hp, HpaI; B, BamHI.

Fig. 2. Efficiency of transformation dependent on (A) electric field voltage and (B) the period of electric impulse: ‘a’ at a constant impulse duration of 2 ms; ‘b’ at a constant field voltage of 1 kV/cm.


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Fig. 3. Effect of the growth phase of algal culture on the transformation efficiency (a) and the growth curve of the CW-15 mutant on mineral nutrient medium (b). Mutant cells sampled at successive stages of the culture growth (10, 15, 25, 30, and 40 h) were treated with electric pulses in the presence of the pCTVHyg plasmid.

and 3) showed that digestion with PstI produces three DNA fragments, 2.7, 1.0, and 0.9 kb in size; two of these fragments (2.7 and 1.0 kb) were homologous to the hybridization probe (Fig. 4, lane 1). Apparently, the C. reinhardtii genome

comprised more than one copy of the hpt sequence (Fig. 4, lanes 2 and 3). In spite of the fact that the pCTVHyg DNA integrated into the nuclear genome of C. reinhardtii was steadily inherited at least within an 8-month period, the HygR trait was manifested in an extremely unstable way. To illustrate this, two transformants (H-l and H-2) plated on the hygromycin-containing medium are discussed. The cell suspension was sampled from a liquid culture at a density of 106 cell/ml and plated on a selective medium with ×l.3 stepwise dilution starting from an inoculum of 104 cells per petri dish 12 cm in diameter. The number of grown HygR colonies in Petri dishes was 0.1 to 1.0% of the total cell number in the inoculum (data are not presented). In addition to viable HygR colonies, numerous tiny and colourless dead colonies were observed. With a microscope, dead microcolonies (4–16 cells) which had completed only one or two sporulation cycles were observed. Similar observations were made immediately after transformation and after 8 months of growth. The second experiment with two HygR clones isolated in the experiment on plating efficiency produced a similar result. All untransformed CW-15 cells (control) perished on the hygromycin-containing medium after 4 or 5 days of growth. 3.4. Comparison of codon usage frequencies in C. reinhardtii nuclear genes and the hpt gene In C. reinhardtii nuclear genes, there is a bias in codon usage: the triplets with A in the third position are met at extremely low frequency [2,3,33]. This type of bias can theoretically preclude the expression of foreign genes in C. reinhardtii cells [2]. Hall et al. [3] calculated the frequency of codon usage for 15 nuclear genes of C. reinhardtii and in nptII [3]. In this study, codon frequencies in the five most representative nuclear genes of C. reinhardtii highlighted by Franco to those in nptII were compared. Franco reported that codons with zero frequency [2] were nevertheless used, a though extremely rarely. The analysis of the hpt sequence [25] using a Microgenie program showed that several codons with terminal A, such as TTA, CTA (Leu), and TCA (Ser), are not used in the hpt gene, whereas other codons with terminal A are used at a frequency ranging from 0.3 to 3.2%. 3.5. Analysis of transient expression of the nptII reporter gene


Fig. 4. Hybridization of total DNA from transformed clones with hpt-specific probes. Total pCTVHyg plasmid DNA restricted with PstI was separated in 0.8% agarose gel, blotted into a nylon membrane and hybridized with a BamHI fragment of the pPCV730 plasmid comprising the coding hpt sequence. Lanes: (1) positive control, (2 and 3) DNA from transformed H-1 and H-2 clones, (4) negative control. The amount of DNA loaded into the gel was 0.1 ␮g for pCTVHyg and 5 ␮g for the initial CW-15 mutant and its transformants.

CW-15 mutant cells were transformed under optimum conditions (1 kV/cm, 2 ms) with pMON129 DNA comprising the recombinant nptII gene, with its coding sequence fused with the regulatory sequences of the nopaline synthase gene [30]. Cells were incubated for 18 h in light, and NPTII activity was assayed in a crude cell extract. NPTII activity in NPTII-producing E. coli cells (Fig. 5, lane 1) and also in all C. reinhardtii cells transformed with the pMON129

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Fig. 5. Analysis of transient NPTII expression. (1) Extract from NPTII-producing E. coli cells (positive control); (2–4) extract from pMON129-transformed cells of C. reinhardtii (three replications); (5) extract from untrasformed CW-15 cells of C. reinhardtii (negative control).

plasmid (Fig. 5, lanes 2–4) vas observed; however, the control cells pulse-treated in the absence of plasmid DNA were devoid of enzyme activity (Fig. 5, lane 5). Currently, the electroporation method is widely used for genetic transformation of microbial, plant, and animal cells [34]. Only one report on C. reinhardtii transformation using the electroporation technique is known to the authors. This was performed at low efficiency (about ten transformants per 106 recipient cells) [35] and due to the widely different conditions used for electroporation, it is difficult to compare the two studies directly. The present data demonstrated that the clones transformed with the hpt gene were unstable, similar to the case of the nptII gene [3]. Yet, using hpt as a selectable marker for C. reinhardtii transformation has some advantages, primarily, the absence of spontaneous HygR mutations. In addition, the evidence presumes that the nptII gene when the latter is integrated into the nuclear genome of algae and inherited in the subsequent generations. Thus, several HygR clones were maintained by regular replating within at least 8 months (about 350 generations) on a hygromycin-free nonselective medium. Another important outcome of this study was the introduction of the CW-15 mutant of C. reinhardtii, with its cells devoid of cell walls, which increased the transformation efficiency by two orders of magnitude.

4. Discussion Currently, the expression of heterologous genes in C. reinhardtii cells is a major issue of genetic engineering studies with this alga; therefore, the use of hpt as a selectable marker, in addition to nptII, seems most promising. Moreover, the selection system employing hygromycin B and the hpt gene is devoid of shortcomings characteristic of the marker of


kanamycin resistance (nptII). This study demonstrated that, first, the CW-15 mutant cells of C. reinhardtii are highly sensitive to hygromycin (10 ␮g/ml) and die by the fourth to fifth day of growth, when plated on soft agar in Petri dishes, and, second, one can disregard spontaneous HygR mutations: not a single HygR colony was observed among the 108 colonies plated on the hygromycin selective medium. Using the electroporation method and selection for the HygR trait in order to transform the CW-15 mutant cells of C. reinhardtii, up to 103 HygR clones were produced per 106 recipient cells. This output is of the same order of magnitude as the efficiency of glass-bead transformation of C. reinhardtii cells [9] and exceeds by two orders of magnitude the efficiency of the electroporation protocol by Brown et al. [35]. Quite unexpectedly, the growth phase in the algal culture notably influenced the efficiency of transformation. This evidence is in contrast with the previous report: Kindle [9] obtained transformants even in stationary phase cells when the rate of DNA synthesis declined. Analysis of HygR clones showed that exogenous DNA when integrated in the nuclear genome of algal cells was inherited without fail in subsequent generations even without selective pressure; i.e. it was mitotically stable. The possibility of autonomous pCTVHyg replication can be excluded. Brown et al. [35] reported that the early-replication bacterial sequences did not support the plasmid in the cytoplasm of C. reinhardtii cells; as a result, plasmid DNA introduced by electroporation rapidly degraded. Even though Hasnain et al. [1] however reported autonomous replication of plasmid DNA carrying the ori gene of the 2 ␮ yeast plasmid under selective conditions [36,37]. Therefore, concerning the stability of heterologous sequences in C. reinhardtii, the present data support the earlier evidence [3,4,17,18,38,39]. However, experiments that assess the efficiency of HygR clones plated on a selective hygromycin medium demonstrated that, in spite of exonous DNA stability, the trait of HygR was maintained extremely inconsistently. Generally speaking, it could be assumed that unstable trait manifestation is governed at any of three levels: DNA, RNA, or protein. In other words, the probable causes of instability include the instability of a foreign gene integrated sequence, factors affecting mRNA transcription and processing, and/or factors influencing the synthesis of a protein product of the gene in question and the further stability of this protein. The possibility that the nucleus and the chloroplast differ when the stability of phenotypic trait manifestation is concerned should also be considered. Currently, there exist data concerning the structural stability of foreign genes, the probability of their transcription, and the activity of their products in C. reinhardtii cells. Several laboratories reported that the uidA, aadA, and nptII gene sequences integrated in the chloroplast genome were consistently inherited through mitoses [17,18,38,39]. Similar evidence concerns the nptII gene integrated in the nuclear genome [3]. Transcription of uidA [17,36] and nptII [38]


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has been reported in chloroplasts. One may exclude at least one probable cause for unstable trait manifestation: apparently, codon usage bias is not a major factor blocking the heterologous gene expression. This suggestion is supported by the comparison of the frequencies of codon usage in nptII [3] and hpt [this paper] to the frequency of codon usage in the nuclear genes of C. reinhardtii. Two foreign proteins, p-glucuronidase (encoded by uidA) and aminoglycoside adeninetransferase (the product of aadA), were active in the chloroplasts [17,18], while NPTII (the product of the nptII gene) was active in the cytoplasm of C. reinhardtii cells [3]. Summing up the available published data, some data obtained both in C. reinhardtii chloroplast and nucleus transformation experiments demonstrate stable integration of heterologous DNA, transcription of the latter, and foreign protein activity. However, the available evidence is not sufficient to make a final conclusion as to the cause of instability of foreign gene manifestation. However, the data obtained with transformed cells do not exclude the possibility that the hpt gene was affected by plasmid DNA rearrangement during long-term cell culturing and that this rearrangement could lead to unstable HygR manifestation. Although transcriptional and translational causes for unstable HygR manifestation are not excluded, the cause for instability may be related to the stability of the gene product, i.e. protein stability to proteolysis. Further studies may provide an equivocal solution to this problem. Genetically determined stability of heterologous proteins has been reported in some organisms. In particular, the inactivation of protease encoded by the E. coli lon gene has been shown to enhance the stability of foreign gene products [40]. Similar mutations have been obtained in Saccharomyces cerevisiae [41]. Presuming that such mutations are possible in C. reinhardtii [42], it seems feasible to attempt to raise the foreign protein stability in this alga through mutagenesis and selection for HygR cells, e.g. by selecting clones manifesting both high HygR and high stability of HygR manifestation. This idea seems especially attractive due to two important advantages of the selection system employed in this study: the high output of HygR transformants and the absence of spontaneous mutations by the HygR trait. Thus, the results obtained in experiments on transformation of chloroplasts and nuclear genes C. reinhardtii testify in favour of long-term stability of integrated heterological DNA, possibility of its transcription and activity of foreign protein. The method of efficient transformation of the cells of C. reinhardtii suggested in the work, provided the stable selective marker has been chosen, can find wide application in biotechnology while obtaining superproducers of foreign proteins.

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