Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer
The prospect of developing large animal models for the study of inherited diseases, such as cystic fibrosis (CF), through somatic cell nuclear transfer (SCNT) has opened up new opportunities for enhancing our understanding of disease pathology and for identifying new therapies. Thus, the development of species-specific in vitro cell systems that will provide broader insight into organ- and cell-type-specific functions relevant to the pathology of the disease is crucial. Studies have been undertaken to establish transformed rabbit airway epithelial cell lines that display differentiated features characteristic of the primary airway epithelium. This study describes the successful establishment and characterization of two SV40-transformed rabbit tracheal epithelial cell lines. These cell lines, 5RTEo- and 9RTEo-, express the CF transmembrane conductance regulator (CFTR) gene, retain epithelial-specific differentiated morphology and show CFTR-based cAMP-dependent Cl− ion transport across the apical membrane of a confluent monolayer. Immunocytochemical analysis indicates the presence of airway cytokeratins and tight-junction proteins in the 9RTEo- cell line after multiple generations. However, the tight junctions appear to diminish in their efficacy in both cell lines after at least 100 generations. Initial SCNT studies with the 9RTEo- cells have revealed that SV40-transformed rabbit airway epithelial donor cells can be used to generate blastocysts. These cell systems provide valuable models for studying the developmental and metabolic modulation of CFTR gene expression and rabbit airway epithelial cell biology.
KeywordsRespiratory airwayEpithelial cell linesSomatic cell nuclear transferSV40 transformationCystic fibrosis transmembrane conductance regulatorBlastocystRabbit (New Zealand White)
A limitation to achieving a better understanding of cystic fibrosis (CF) pathology and to the development of novel therapies for CF has been the ineffectiveness of the CF mouse model (Grubb and Boucher 1999). The recent development of the CF pig (Rogers et al. 2008b, 2008c) and ferret (Li et al. 2006; Sun et al. 2008) have provided additional animal models that can potentially be used to elucidate CF pathology further and to develop therapies for CF. The rabbit is also an excellent candidate for an animal model of CF, because of the anatomical, genetic and biochemical similarities between the rabbit and the human and because of their gestation time and reproductive ability (Bosze et al. 2003; Chen et al. 2001; Diamond et al. 1991; Fan and Watanabe 2003; Shiffman et al. 1983). Both the phylogenetic nature of the rabbit and its physiology and anatomy make the rabbit a relevant model for the biochemical, molecular and physiological characterization of CF pathology and for the development of CF therapies (Vuillaumier et al. 1997; Zeitlin et al. 1992). Since no viable embryonic stem (ES) cell systems exist that have been established for the development of transgenic animals and germline transfer outside of the mouse, the generation of large animal models is dependent on cloning technology and somatic cell nuclear transfer (SCNT; Challah-Jacques et al. 2003; Kasinathan et al. 2001; Matsuda et al. 2002).
Verification that the same or similar biochemical and molecular pathways are in place in the animal model to be developed for CF requires that in vitro cell systems relevant to CF are developed. Whereas primary airway epithelial cell systems have been a powerful tool in this regard (Gruenert et al. 1990; Widdicombe et al. 1985), their limited lifespan attributable to senescence or terminal differentiation (Hayflick 1974, 1998) has been an impediment to the analysis of biochemical pathways and the development of CF therapies. This limitation for airway epithelial cells has however been overcome through the transformation and/or immortalization of cultured primary cells (Cozens et al. 1992a; de Semir et al. 2008; Gruenert 1987; Gruenert et al. 1988, 1995, 2004). The transformation and immortalization of airway epithelial cells has been integral in the study of airway disease and for expanding our understanding of metabolic and genetic mechanisms underlying airway epithelial cell functions associated with CF pathology.
Once established, the transformed cells exhibit an enhanced potential to proliferate. Ultimately, the transformed cultures generally but not always, go through a period of “crisis” during which the cells either die or become immortalized established cell lines (de Semir et al. 2008; Gruenert 1987; Gruenert et al. 1995). Most immortalized airway epithelial cell lines have been established by using simian virus 40 (SV40)-based systems (Gruenert et al. 2004). One approach that has been proven to be particularly effective has been the transfection of the pSVori- plasmid that contains a replication defective SV40 genome (Gruenert et al. 1988; Small et al. 1982). Previous studies that have focused on the transformation, immortalization and characterization of human airway epithelial cells (Cozens et al. 1992a, 1994; de Semir et al. 2008; Ehrhardt et al. 2006; Goncz et al. 1999; Gruenert 1987; Gruenert et al. 1988, 1995, 2004) clearly demonstrate that the development of large animal models for CF will benefit from in vitro cell systems with regard to the effective validation of the biochemical features of the animal model. The present study has evaluated the transformation/immortalization of rabbit airway epithelial cells with the pSVori- plasmid that contains an SV40 viral genome defective in the SV40 origin of replication (Small et al. 1982). The transformed/immortalized rabbit tracheal epithelial (RTE) cells have been characterized karyotypically, immunocytochemically and in terms of airway epithelial cell gene expression, including CFTR, keratin and zonula occludens factor 1 (ZO-1) and functional Cl− ion transport. The analyses of these features suggest that the cells can be used as an in vitro model of the airway epithelium and are potentially useful for the development of therapies for CF. In addition to the generation of in vitro cell systems from primary cells, SCNT used in the development of transgenic animal models has provided another potential means for generating lineage-specific immortalized cell lines. Several studies have evaluated the potential of primary epithelial cells to act as donor cells for SCNT for the production of a viable embryo or blastocysts (Gong et al. 2004; Kishigami and Wakayama 2009). However, no studies have evaluated the potential of using transformed cells to generate viable blastocysts. This report demonstrates that transformed aneuploid rabbit airway epithelial cells will form blastocyts at frequencies higher than those observed by using primary cells (Du et al. 1995).
Materials and methods
Isolation of RTE cells
An excised trachea from a female New Zealand White rabbit (Oryctolagus cuniculus; Evergen Biotechnologies) was rinsed four times with phosphate-buffered saline (PBS; Invitrogen, Carlsbad, Calif., USA) containing 5 mM dithiothreitol and twice in PBS alone. The tissue was incubated overnight at 4°C in PBS containing 1 mg/ml pronase (Roche, Mannheim, Germany). The following day, epithelial cells were collected by flushing the tracheal lumen with the 4°C PBS-pronase solution. The cells were then pelleted by centrifugation. The cell pellet was washed once with ice-cold PBS containing 10% fetal bovine serum (FBS) to neutralize the pronase. The cells were then resuspended in modified serum-free LHC-8e medium (MLHC8e) and plated on tissue culture plastic coated with a solution containing 10 mg/ml human fibronectin (BD laboratories, Franklin Lakes, N.J., USA), 30 mg/ml Vitrogen (Cohesion Technologies, Palo Alto, Calif., USA) and 100 mg/ml bovine serum albumin (BSA) Fraction V (FN/V/BSA) in LHC basal medium (Invitrogen; Gruenert et al. 1990). Cultures were routinely grown in a humidified atmosphere under 5% CO2 at 37°C.
RTE cell transformation
The passage-2 primary RTE cells were grown in pre-coated 100-mm dishes to 70%-80% confluence, trypsinized and then electroporated/nucleofected with the pSVori- plasmid by using the T-07 program and the Basic Nucleofector Kit for primary mammalian epithelial cells (Amaxa, Gaithersburg, Md., USA) according to the manufacturer’s instructions as described previously (Maurisse et al. 2010). After electroporation, the transfected cells were immediately plated onto coated 100-mm culture dishes and grown to confluence in MLHC8e, with routine medium changes every other day. The isolation of the viable proliferating transformants was straightforward, because the nontransformed airway epithelial cells undergo terminal squamous differentiation and senescence (de Semir et al. 2008; Gruenert 1987; Gruenert et al. 1988, 1995). Colonies of transformed cells were isolated by using cloning rings and expanded on coated 100-mm dishes in Eagle’s Minimum Essential Medium (MEM) with Earle’s salts and supplemented with penicillin/streptomycin, glutamine (200 mM) and 10% FBS for further characterization. Although, multiple clones were isolated, clones 5RTEo- and 9RTEo- were initially selected for further analysis.
Immortalized human bronchial epithelial cells
The clones were compared with a well-characterized immortalized human airway epithelial cell line, 16HBE14o- (Cozens et al. 1994) in terms of their airway-epithelial-specific features. As above, these cells were routinely grown on the same pre-coated dishes and/or flasks in MEM supplemented with antibiotics, glutamine and 10% FBS under 5% CO2.
Densitometric analysis of amplification of CFTR mRNA by reverse transcription with the polymerase chain reaction
Cytoplasmic RNA was extracted from the primary and immortalized cells by using the RNeasy mini kit (Qiagen, Valencia, Calif., USA) and then treated with DNase I (8.3 U/μg RNA, Roche, Mannheim, Germany) for 1 h at 37°C. RNA (3 μg) was heated to 70°C for 10 min and then reverse-transcribed by using random hexamers (1 μg), oligo dT (1 μg) and Superscript II RNase H- reverse transcriptase (Invitrogen). CFTR mRNA from this reverse transcription was analyzed by standard polymerase chain reaction (RT-PCR) with rabbit-specific primers r1278F: 5’-ggctggatctactggtgctg-3’ (exon 9) and r1830R: 5’-aaaatccggccgtagacttt-3’ (exon 11). PCR amplification was carried out in a 50-μl solution containing 5 μl 10× Taq Platinum polymerase buffer, 1.5 μl MgCl2 (10 mM), 1 μl dNTPs (10 mM), 0.5 μl Taq Platinum polymerase (Invitrogen), 1 μl primers of r1278F and r1830R (15 μM) and 2 μl cDNA. The conditions for the amplification were as follows: an initial denaturation for 3 min at 94°C, followed by 32 cycles of: denaturation at 94°C for 30 s, annealing at 62°C for 30 s and extension at 72°C for 50 s, with a final extension of 7 min at 72°C. The 552-bp amplicon was sized on an E-Gel pre-cast 2% agarose gel (Invitrogen). Bands were analyzed densitometrically by using ImajeJ software.
Passage-2 (P2) primary and transformed cells (P1.3) were grown on well slides (Lab-Tek, Thermo Fisher Scientific, Rochester, N.Y., USA) coated with the FN/V/BSA solution. After being washed, fixed and dried, the cells on the slides were rehydrated in PBS and stained for immunofluorescence analysis. The primary antibodies used were: mouse monoclonal antibody Pab 101 (anti-SV40 T Ag; Santa Cruz Biotechnology, Santa Cruz, Calif., USA) at 1:50 dilution; AE1/AE3 (anti-cytokeratin) mouse monoclonal anti-human cytokeratin 1,2,3,4,5,6,7,10,13,14,15,16,19 antibody (DakoCytomation, Carpinteria, Calif., USA) at 1:10 dilution; DC-10 mouse monoclonal anti-cytokeratin 18 antibody (Santa Cruz Biotechnology) at 1:200 dilution; H-300 rabbit polyclonal anti-zonula occludens-1 (ZO-1) antibody (Santa Cruz Biotechnology) at 1:5 dilution. The secondary antibody, namely bovine anti-rabbit IgG conjugated to fluorescein isothiocyanate (Santa Cruz Biotechnology), was used at a 1:100 dilution. All the antibodies listed above were incubated with the cells for 30 min at 37°C and the cells were then washed three times with PBS.
The presence of the SV40 large T antigen indicated that the cells were transformed/immortalized by the pSVori- plasmid. Staining with the cytokeratin 18 (K-18) or the AE1/AE3 antibodies indicated the presence of an airway epithelial cytoskeletal structure. ZO-1 as a component of the zonula occludens was an indicator of intercellular tight junctions.
Primary and immortalized RTE cells were grown to 70%-80% confluence in MLHC8e medium or MEM, respectively, on coated 100-mm dishes. The cultures were incubated overnight at 37°C in the presence of 0.04 mg/ml colcemid (Invitrogen). After a 25-min exposure to hypotonic 75 mM KCl (Sigma, St Louis, Mo., USA), the cells were removed from the dish by using a cell scraper, centrifuged and fixed in Carnoy’s solution (3:1 methanol:acetic acid). The cells were resuspended and spread dropwise on frozen glass slides. They were then air-dried and stained with Giemsa (4% in 10× PBS; Sigma) for 7 min. The 5RTEo- and 9RTEo- cells were treated with 0.025% trypsin in 10× PBS for 2 s to obtain a G-banding pattern. Stained slides were viewed without a coverslip under a Nikon microscope at 100× magnification. The average chromosome number was determined by counting chromosomes on microphotographs by using 3, 9 and 20 metaphase spreads for the 5RTEo-, RTE and the 9RTEo- cell lines, respectively.
Stock cultures of confluent transformed/immortalized RTE cells (5RTEo-P1.12 and 9RTEo-P1.5) were trypsinized, resuspended in fresh MEM and plated on coated (as above) 12-mm clear polyester Snapwell inserts (Corning, Lowell, Mass., USA). Approximately 105 cells were plated onto each insert and grown for 9–11 days. Snapwell inserts were mounted via sliders into Easy Mount Ussing chambers (Physiologic Instruments, San Diego, Calif., USA) and short circuit current (ISC) measurements were performed as previously described for CFBE41o- monolayers (Illek et al. 2010). Briefly, a typical four-electrode voltage clamp (VCC MC6, Physiologic Instruments, San Diego, Calif., USA) was connected via Ag/AgCl electrodes (World Precision Instruments, Sarasota, Fla., USA) to the solutions through 3% Agar bridges containing 1 M KCl. ISC was continuously recorded at 5 Hz by an analog-to-digital board (DATAQ Instruments, Akron, Ohio, USA) and at 60 s intervals, the transepithelial voltage was clamped for 1 s from 0 to 1 mV to monitor transepithelial resistance (Rt) by using Ohm’s law. A basolateral-to-apical Cl- gradient was established to increase the electrochemical driving force for Cl− secretion across the apical membrane and the transepithelial ISC measured under these conditions was termed ICl. The basolateral solution was composed of 120 mM NaCl, 25 mM NaHCO3, 5 mM KCl, 1.2 mM NaH2PO4, 5.6 mM glucose, 1.0 mM CaCl2 and 1.2 mM MgCl2), whereas the apical Cl-free solution was composed of 120 mM Na-gluconate, 20 mM NaHCO3, 5 mM KHCO3, 1.2 mM NaH2PO4, 5.6 mM glucose, 2.5 mM Ca(gluconate)2 and 1.2 mM MgSO4. Both chamber compartments were separately perfused with 5 ml of each solution at 37°C, respectively and gassed with 5% CO2 in air to give a pH of 7.4. Positive currents were defined as the movement of anions in the basolateral-to-apical direction. To determine the magnitude of transcellular Cl− ion movement, CFTR blockers (CFTR-inh172, GlyH-101) were used at the end of each experiment to block Cl− exit across apical CFTR Cl channels. ICl and the change in forskolin-stimulated Cl currents (∆ICl) were compared between clones 5RTEo- and 9RTEo-.
Oocyte collection and SCNT
Rabbit oocytes were collected and prepared as described previously (Du et al. 2009). All micromanipulations were carried out by using a standard protocol (Du et al. 2006). Individual tracheal epithelial donor cell nuclei (9RTEo- P1.6) were transferred into the perivitelline space of an enucleated oocyte. Cell fusion and monitoring of the success of SCNT was performed as reported previously (Du et al. 2009).
Two cells lines were generated, namely 9RTEo- and 5RTEo-, after transfection of primary RTE cells with the pSVori- plasmid (de Semir et al. 2008; Gruenert 1987; Gruenert et al. 1988, 1995). Individual colonies were isolated and grown until confluent.
Transepithelial ion transport
Transepithelial electrical parameters of the rabbit tracheal epithelial cell lines (P passage). All measurements were made in the presence of serosal-to-mucosal Cl− gradients. Chloride currents (ICl in μA/cm2) and transepithelial resistances (RT in Ωcm2) were measured before treatment (Unstimulated), after the addition of forskolin (Forskolin) and following the addition of the CFTR inhibitor (GlyH-101). ΔICl = ICl (after) − ICl (before) treatment. *Measurements significantly different from unstimulated values by one way analysis of variance. Values are the mean ± SD (n number of experiments)
9RTEo- (P 1.5)
5RTEo- (P 1.12)
In vitro development of rabbit embryos by SCNT
In vitro development of rabbit cloned embryos from 9RTEo- cells. Somatic cell nuclear transfer (SCNT) of 9RTEo- nuclei into rabbit oocytes. The nuclei transferred (NT) were monitored in terms of their developmental progression to the blastocyst stage. The proportion of SCNT oocytes progressing to each stage is indicated as a percent of the total
Number of NT assays
Four-cell stage (%)
Blastocyst day 5 (%)
The present study describes the transformation of primary RTE cells with the pSVori- plasmid (Cozens et al. 1992a, 1992b, 1994; de Semir et al. 2008; Gruenert et al. 1988, 1995, 2004; Small et al. 1982). Clones of transformed cells have been characterized with respect to their epithelial-specific features and their karyotype. Immunocytochemical characterization has indicated that the transformed cells express the SV40 large T antigen and cytokeratin, a hallmark of epithelial cells. The primary and transformed cells also possess ZO-1 indicating that they have maintained their epithelial-specific junctional complexes (Cozens et al. 1992a, 1994; de Semir et al. 2008; Gruenert 1987; Gruenert et al. 1988, 1995, 2004). Although the transformed cells are aneuploid, they also retain the epithelial-specific expression of CFTR. Furthermore, when nuclei from the transformed cells are used as donor nuclei for SCNT, they are effective in generating multicellular blastocysts.
Over the last two decades, a number of rabbit cell lines from diverse tissue origins have been generated after transfection of the SV40 early genes (Burns et al. 1996; Nachtigal et al. 1990; Taub et al. 2002; Thenet et al. 1992) and numerous airway epithelial cell lines have been developed to study various airway diseases such as CF (Gruenert et al. 2004). An aneuploid karyotype is generally associated with cell transformation (de Semir et al. 2008; Gruenert 1987; Gruenert et al. 1995) and has been observed in transformed human airway epithelial cells and in SV40-transformed rabbit kidney or corneal epithelial cells (Kang et al. 2001; MacDonald et al. 1991). Kang et al. (2001) have observed a mean of 72.8 chromosomes per cell in their SV40 immortalized cells, a value that is consistent with the mean number of chromosomes per cell observed in this study. Another study involving the use of SV40 large T antigen transformation of an RTE cell line has evaluated squamous cell differentiation (Lotan et al. 1992); however, these cells have not been extensively assessed for tight junctions, cytokeratins, or karyotype. Extensive Ussing chamber analysis of ion transport has shown that the 9RTEo- and the 5RTEo- cells maintained cAMP-dependent Cl− ion transport and indicates that the cells at early passages maintain their tight junctions and CFTR-dependent Cl− transport. At later passages, the clones that we have assayed begin to lose transepithelial resistance, even though they retain their CFTR-dependent Cl− ion transport. Apparently, some cells within the population lose their ability to retain tight junctions, whereas others do not, thus resulting a “porous” monolayer. To circumvent the loss of tight junctions, additional clones generated from the initial transformation can be analyzed. Alternatively, 9RTEo- and 5RTEo- cell lines can be subcloned to select for those cell colonies that retain their tight junctions.
The study of diseases such as CF has relied primarily on murine models (Grubb and Boucher 1999; Guilbault et al. 2007). However, the mouse is not an ideal model for the study of CF disease pathology, in particular, airway disease. Therefore, the development of (1) alternative animal models to study the progression of CF disease and (2) cell systems to elucidate the molecular and biochemical mechanisms underlying the cellular dysfunction associated with CF is crucial. The recent development of the CF pig (Rogers et al. 2008a, 2008b, 2008c) and CF ferret (Li et al. 2006; Sun et al. 2008) has added to the repertoire of animal model systems. However, long-term cell culture systems have not been developed for either of these animal models.
In addition to the pig and ferret, the rabbit is an attractive candidate as an animal model to gain a better understanding of the mechanisms that underlie CF pathophysiology. The rabbit not only is anatomically relevant to the human but also has a short gestation period (28–31 days), is relatively low maintenance and is easy to handle. Whereas SCNT and animal cloning has become routine (Li et al. 2009; Matsuda et al. 2002), rabbit cloning is considered difficult (Yang et al. 2007). To date, only four reported cases worldwide have achieved success in SCNT in the rabbit. However, the favorable outcome of these studies demonstrates the feasibility of rabbit cloning by using cumulus cells (Chesne et al. 2002; Du et al. 2009), stem cells (Challah-Jacques et al. 2003) and adult fibroblasts (Shi et al. 2008) as nuclei donors.
In a previous study, a comparison of primary cultures of cumulus cells with fetal fibroblasts for their efficiency in SCNT has revealed that the donor cell type influences the efficiency of rabbit cloning (Cervera and Garcia-Ximenez 2003). Whereas fetal fibroblasts are more effective at fusion than the cumulus cells (67% versus 45%), in vitro development to the morula (cumulus cells at 41% versus fetal fibroblasts at 14%) and the blastocyst (cumulus cells at 27% versus fetal fibroblasts at 3%) stages is different between cell types. The 66% fusion efficiency observed on using the pSVori- transformed RTE cells (Table 2) is comparable with that observed for the fetal fibroblasts. However, the 28.8% and 25.7% observed with regard to progress to the morula and blastocyst stages, respectively, are similar to the pecentage observed when cumulus cells are used as donors (Cervera and Garcia-Ximenez 2003), i.e., almost all the embryos that reach the morula stage develop into blastocysts (17 of 19).
Few reports have assessed the feasibility of SCNT and cloning by using immortalized cell lines as nuclei donors (Cui et al. 2003; Zakhartchenko et al. 1999). In species other than rabbit, either a failure to generate blastocysts from a spontaneously immortalized bovine mammary gland epithelial cell line (Zakhartchenko et al. 1999) or an inability to generate embryos from a telomerase-immortalized sheep fibroblast (Cui et al. 2003) has been experienced. This is the first report demonstrating that blastocysts can be obtained from a donor cell line that has been transformed with an origin of replication-defective SV40 plasmid.
Two groups have shown that rabbit embryonic stem (rES) cell lines can be derived from preimplantational embryos (Graves and Moreadith 1993) or from non-immortalized SCNT blastocysts (Fang et al. 2006). These results and the finding that various cell types (fibroblasts, ES cells, or trophoblasts) can be generated from blastocysts of species other than rabbit (Gomez et al. 2006; Hatoya et al. 2006; Li et al. 2003; Shimada et al. 2001) provide the novel possibility of using the blastocysts generated from these immortalized cell nuclear donors to develop immortal in vitro cell systems with cell-type-specific properties that not only can give insights into human diseases but also facilitate the development of novel therapies.
We thank Dr. Michael Yezzi, Janet Nguyen, Judy Cheung and Lindsay Juarez for their technical assistance and Drs. A. Abdolmohammadi and B. Bedayat for their input and commentary.