Introduction

Pigs are the most economically important livestock, but pig cell lines useful for physiological studies, virus isolation and/or vaccine development are limited in cell banks. One of the many reasons for the limited number of pig cell lines is that it is difficult to establish such cell lines (Donai et al. 2014). The most common method for generating animal cell lines is to transform the primary cells with viral or cellular oncogenes. However, the cell lines generated with these approaches are invariably transformed, genomically unstable and/or phenotypically different from their normal counterparts (Ouellette et al. 2000). More recently, human telomerase reverse transcriptase (hTERT) has been used to immortalize animal cells. Unlike oncogene transformation, TERT-mediated immortalization does not cause cellular transformation and genomic instability, which represents a major improvement over the use of oncogenes for generating animal cell lines (Morales et al. 1999; Jiang et al. 1999; Ouellette et al. 2000).

Several pig cell lines have been generated by hTERT-mediated immortalization using plasmid- or viral vector-based methods (Cao et al. 2011; Hong et al. 2007; Ibi et al. 2007; Kuruvilla et al. 2007; KwaK et al. 2006; Pan et al. 2010; Sagong et al. 2012; Uebing-Czipura et al. 2008). The plasmid-mediated gene transfer is inefficient and thus requires the use of cytotoxic drugs for the selection of desired cell clones with long culture times. Viral vectors (e.g., lentiviral vectors) have the advantages of high gene transfer efficiency and stable gene expression (O’Reilly et al. 2012; Mátrai et al. 2010). However, the genomic integration of lentiviral DNA is strongly biased toward transcriptionally active loci, leading to an increased risk of insertional mutagenesis and/or biosafety concerns (Staunstrup et al. 2009). On the other hand, transposons have been considered as natural, non-viral gene delivery vehicles capable of efficient genomic insertion (Izsvák et al. 2009). In addition, transposons use a precise recombinase-mediated mechanism to mediate gene integration, providing long-term gene expression with improved simplicity and safety (Izsvák et al. 2009; Belay et al. 2011). In this report, we present a new method for immortalization of pig cells by using Sleeping Beauty (SB) transposon system-mediated expression of porcine TERT (pTERT).

Materials and methods

Preparation of primary pig fibroblast cells

A 2-week-old Chinese local breed Jangquhai pig was provided by the National Pig Breed Conservation Center (Taizhou, China). Primary pig fibroblast cells (PFCs) were prepared from the pig kidney as preciously described (Onishi et al. 2000) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen Life Technologies, Carlsbad, CA, USA). Cells were incubated in a 37 °C incubator supplemented with 5 % CO2 and non-PFC cells were removed by 3 cycles of incubation for 20 min according to their slower adhesion onto flask surface.

PCR for cloning porcine elongation factor-1a promoter

Genomic DNA was extracted from PK-15 cells (ATCC, Manassas, VA, USA) using Genomic DNA Extraction Kit (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s protocol. Porcine elongation factor 1a (pEF-1α) promoter was cloned by PCR using Ex Taq™ Polymerase (TaKaRa Biotechnology, Dalian, China) and the primer pair in Table 1. The PCR was carried out at 95 °C for 5 min, 30 cycles of 94 °C for 30 s, 59 °C for 30 s and 72 °C for 30 s, and a final extension at 72 °C for 10 min. The PCR product was subcloned into pMD18-T vector (TaKaRa Biotechnology, Dalian, China) for sequencing.

Table 1 The PCR primers used in this study
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RT-PCR for pTERT cDNA cloning

Cellular RNA was extracted from PK-15 cells using RNAiso Plus (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s manual. pTERT cDNA was cloned by RT-PCR using PrimeScript™ RT-PCR Kit (TaKaRa Biotechnology, Dalian, China) and the primer pair in Table 1. The PCR was carried out at 95 °C for 5 min; 2 cycles of 94 °C for 30 s, 64 °C for 30 s and 72 °C for 3 min; 2 cycles of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 3 min; 2 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 3 min; 26 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 3 min, and a final extension at 72 °C for 10 min. The PCR product was subcloned into pMD18-T vector for sequencing.

SB transposon vector construction

The poly(A) signal of bovine growth hormone (BGH) gene was excised from pcDNA3 vector (Invitrogen Life Technologies, Carlsbad, CA, USA) by restriction digestion and inserted into SB transposon vector pT2/HB (Addgene, Cambridge, MA, USA) as a XbaI/BglII segment. The pEF-1α promoter was excised from pMD18-T vector and cloned into the transposon vector as an EcoRV/NotI segment. The modified SB transposon vector was called pTEG (Fig. 1). The coding sequence for green fluorescent protein (GFP) was amplified from pEGFP-N1 vector using the primer pair in Table 1. The PCR product was inserted into the pTEG vector and the resultant vector pTEG-GFP was used for validation purpose. The pTERT cDNA was subcloned into the pTEG vector and the resultant vector pTEG-TERT was used for generating pig cell lines.

Fig. 1
figure 1

The schematic structure of modified SB transposon vector pTEG. IR/DR, left and right terminal inverse repeats of SB transposon; PEF-1α, porcine elongation factor-1a promoter; MCS, multiple cloning sites; BGH pA, poly(A) signal of bovine growth hormone; SB integration; SB transposase-integrated expression cassette

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Cell transfection

The SB transposon vectors pTEG-GFP and pTEG-TERT, as well as the transposase vector pSB16 which encodes SB transposase (Addgene, Cambridge, MA, USA), were prepared from the transformed DH5α E. coli cultures using EndoFree Plasmid Maxi Kit (Qiagen, Shanghai, China). Primary PFCs were seeded at 105 cells/well on 6-well plates, grown to 80 % confluency, and transfected with the transposase vector (0.6 µg/well) and/or transposon vector (3.4 µg/well) using Lipofectamine™ 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instruction.

Validation of SB transposon system

Primary PFCs were transfected with the transposase vector and/or transposon vector pTEG-GFP, and passed for 7 generations before fluorescent microscopy for GFP-positive cells. The integration efficiencies for random (without transposase) and recombinase-mediated (with transposase) gene transfer were calculated according to the percentages of GFP-positive cell numbers.

Cell cloning and identification

Primary PFCs were transfected with the transposase vector and transposon vector pTEG-TERT as described. On day 2 after transfection, the confluent cells were passed for 1 generation and cloned by 3 cycles of dilution cloning as previously described (Freshney 2010). The genomic DNA was extract from each cell clone using Genomic DNA Extraction Kit (TaKaRa Biotechnology, Dalian, China) and amplified for pTERT expression cassette or ampicillin-resistant gene (ampR) on the vector by PCR using the primer pairs in Table 1. The PCR was carried out at 95 °C for 5 min, 25 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, and a final extension at 72 °C for 5 min. The PCR products were analyzed by 1.2 % agarose gel electrophoresis. One cell clone containing pTERT expression cassette (ampR −), but not intact transposon vector (ampR +), was passed for different generations and further characterization.

Telomerase assay

Both PFC cell line and the primary cells at different generations were seeded on 6-well plates and grown to 80 % confluency. The cells were trypsinized, diluted to 106/ml with PBS (pH7.4) and lyzed by 3 cycles of freeze/thawing. The telomerase activities in the centrifuged cell extracts were measured using Porcine Telomerase ELISA Kit (Jianglaibio, Shanghai, China) according to the manufacturer’s instruction. The primary PFC (passage 3) lysate was heated for 15 min at 85 °C and used as the negative control.

Growth curve plotting, population doubling time and cloning efficiency calculation

Both PFC cell line and the primary cells at different generations were seeded in triplicates at 104cells/well on 4-well plates. On day 2 after plating, the cells were trypsinized daily for growth curve plotting and population doubling time calculation as previously described (Hong et al. 2007). At the same times, the cells were seeded in triplicates at 1000, 500, 200, 100 or 50 cells/well on 6-well plates and grown for 2 weeks for cloning efficiency calculation as previously described (Hong et al. 2007).

RT-PCR for detection of marker gene expression

The cellular RNA was extracted from PFC cell line (passage 40), primary PFCs (passage 3) or PK-15 cells using RNAiso Plus and the cDNAs were generated using PrimeScript™ RT-PCR Kit (TaKaRa Biotechnology, Dalian, China). The primer pairs for PCR amplification of porcine CD29, vimentin (Vim), CK18 and p53 transcripts are listed in Table 1. The PCR was carried out at 95 °C for 5 min, 25 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, and a final extension at 72 °C for 5 min. The PCR products were analyzed by 1.2 % agarose gel electrophoresis.

Cell cycle analysis

Both PFC cell line (passage 40) and the primary cells (passage 3) were seeded at 105cells/well on 6-well plates and grown to 80 % confluency. The cells were trypsinized, fixed overnight at 4 °C with 70 % ethanol and stained with 50 µg/ml propidium iodide for DNA content measurement on BD FACSARIA III (BD Biosciences, Franklin Lakes, NJ, USA) as previously described (Hang and Fox 2004).

Soft agar clone formation assay

The trypsinized PFC cell line (passage 40) and primary PFCs (passage 3), as well as mouse myeloma cell line Sp2/0 (Santa Cruz Biotechnology, Paso Robles, CA, USA), were diluted to 106 cells/mL with 2 × DMEM containing 20 % FBS, mixed with equal volumes of 0.7 % low melting agarose solution (40 °C) and incubated in 35-mm dishes. After incubation for 2 weeks, the cell colonies were observed under light microscope as previously described (Akagi et al. 2003).

Statistical analysis

The data of telomerase activities and cell numbers of PFC cell line and primary cells were expressed as mean ± SD (n = 3) and statistically analyzed using Student’s t test.

Results

Modification of SB transposon vector

Eukaryotic EF-1α promoters have a robust transcriptional activity in a variety of cell types (Norrman et al. 2010). However, the transcriptional activity of pEF-1α promoter has not been tested. To modify the SB transposon vector for stable pTERT expression in pig cells, we cloned pEF-1α promoter from PK-15 cells by PCR. Sequence analysis showed that the cloned promoter was almost identical to the published sequence (GenBank: FM995601) with only one C to T transition at position 20. The pEF-1α promoter, as well as the poly(A) signal of BGH gene, was subcloned into the SB transposon vector and the modified vector was called pTEG (Fig. 1). To validate the SB transposon system in pig cells, primary PFCs were transfected with the transposon vector pTEG-GFP alone or co-transfected with the transposase vector pSB16. At 24 h after transfection, similar numbers of GFP-positive cells were revealed in the cell cultures transfected with pTEG-GFP with or without pSB16. After passage for 7 generations (P7), however, much more GFP-positive cells remained in the cell culture co-transfected with pTEG-GFP and pSB16, but not in the cell culture transfected with pTEG-GFP alone (Fig. 2).

Fig. 2
figure 2

Validation of the SB transposon system in pig cells. Primary PFCs were transfected with the transposon vector pTEG-GFP or co-transfected with the transposon vector pTEG-GFP and the transposase vector pSB16 which encodes SB transposase. The cell cultures were observed under fluorescent microscope for GFP-positive cells before (P0) and after passage for 7 generations (P7)

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Cloning and expression of pTERT cDNA

Several pig cell lines have been generated by hTERT-mediated immortalization. Although the cDNA sequence for pTERT is available in GenBank, it has not been tested experimentally whether the cDNA can encode a functional enzyme. Therefore, we cloned pTERT cDNA from PK-15 cells by RT-PCR. Sequence analysis showed that the cloned cDNA was 99.6 % identical to the published sequence (GenBank: NM-001244300) with only two G to A transitions at positions 548 and 582, and a TGCT to GCTG change from positions 2169 to 2172. The cDNA was subcloned into the pTEG vector and transfected into primary PFCs. Enzymatic assay showed that more than 200 ng/L telomerase activity was over expressed in the vector-transfected cells.

Selection and identification of PFC clones

To obtain the cell clones containing pTERT expression cassette only, primary PFCs were transfected with the SB transposon system and cloned by three cycles of dilution cloning. After one generation passage, the genomic DNA was extracted from each cell clone and analyzed for pTERT expression cassette or ampR gene on the vector by PCR. Among ten cell clones identified, two clones harbored the pTERT expression cassette only (Fig. 3) and one of them was selected for generation of a PFC cell line.

Fig. 3
figure 3

Identification of cell clones by PCR. Primary PFCs were co-transfected with transposon vector pTEG-TERT and the transposase vector pSB16. After 1 generation passage, the cells were cloned by three cycles of dilution cloning and the genomic DNA was extracted from cell clones for detection of pTERT expression cassette or ampR on the transposon vector. Random integration indicates the cell clone with randomly integrated transposon vector and SB integration indicates another cell clone with transposase-integrated TERT expression cassette

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Ectopic pTERT expression in PFC cell line

Two different methods were used to test the ectopic pTERT expression in PFC cell line using the primary cells as the control. First, cellular RNA was isolated at different passages and analyzed for pTERT transcription by RT-PCR. An expected 236-nt pTERT transcript segment was detected in the cell line at passage 0, 10, 20, 30 or 40 (Fig. 4a), whereas the same transcript was detected in the primary cells before passage 5 (Fig. 4b). Then, cell extracts were prepared at different passages and analyzed for telomerase activity using telomerase-inactivated PFC lysate as the negative control. An averaged 410 ng/L telomerase activity was detected in PFC cell line before passage, which was decreased slightly by passage 10 and remained at the similar level by passage 40 (Fig. 4a). In contrast, only 200 ng/L telomerase activity was detected in the primary cells before passage 5, which was undetectable by passage 10 (Fig. 4b).

Fig. 4
figure 4

Detection of ectopic pTERT expression in PFCs. Top panels, cellular RNA was extracted from PFC cell line (a) or the primary cells (b) at different passages (P) and amplified for pTERT transcript by RT-PCR. Bottom panels, cell extracts were prepared at different passages and assayed for telomerase activity by ELISA using heat-activated cell extract as the negative control

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Growth properties of PFC cell line

The PFC cell line kept active proliferation by passage 40, whereas the primary cells have undergone senescence as early as passage 10. Light microscopy showed that the PFC cell line at different passages kept the fibroblast morphology of primary cells (Fig. 5a). The cell line at different passages exhibited contact inhibition in FBS-containing medium on day 5 after cultivation. Growth curve plotting showed that the PFC cell line at passage 10, 20, 30 or 40 had a growth curve similar to the primary cells, reaching the stationary phase by day 6 after plating (Fig. 5b). However, the cell numbers of PFC cell line at stationary phase were significantly higher than those of the primary cells, especially after passage 20 (Fig. 5b). The PFC cell line had a slightly shorter population doubling time and higher cloning efficiency than the primary cells, especially after passage 20 (Table 2).

Fig. 5
figure 5

Morphology and growth curve of PFC cell line. a PFC cell line and the primary cells were observed under light microscope (×45). b Cell numbers of PFC cell line or the primary cells at different passages were plotted against the culture times

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Table 2 Population doubling times and cloning efficiencies of PFCs
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Marker gene expression of PFC cell line

To confirm the identity of PFC cell line, cellular RNA was extracted and analyzed for marker gene expression using primary and PK-15 cells as the controls. As expected, the transcript for porcine mesenchymal stromal cell marker CD29 was detected in all of the three cell types tested (Fig. 6), whereas the transcript for porcine mesenchymal cell marker vimentin (Vim) was detected in PFC cell line and the primary cells (Fig. 6a, b), but not in PK-15 cells (Fig. 6c). On the other hand, the transcript for porcine epithelial cell marker CK18 was detected in PK-15 cells (Fig. 6c), but not in PFC cell line and the primary cells (Fig. 6a, b). Additionally, the transcript for porcine p53 was also detected in the three cell types (Fig. 6).

Fig. 6
figure 6

Marker gene expression of PFC cell line. Cellular RNA was extracted from primary PFCs (a), PFC cell line (b) or PK-15 cells (c), and amplified for the transcript of porcine CD29, vimentin (Vim) or CK18 by RT-PCR

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Cell cycle distribution of PFC cell line

Flow cytometry was used to analyze the cell cycle distribution of PFC line using the primary cells as the control. Histogram plots showed no significant difference in cell cycle distribution between PFC line and the primary cells under normal culture conditions (Fig. 7).

Fig. 7
figure 7

Cell cycle distribution of PFC cell line. Primary PFCs (a) or PFC cell line (b) were grown to 80 % confluent growth and stained with propidium iodide for DNA content measurement by flow cytometry

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Clone formation of PFC cell line

PFC cell line was plated on soft agar plates using the primary cells and mouse myeloma cell line Sp2/0 as the controls. After incubation for 2 weeks, no cell colony was observed on the agar surface plated with the PFC cell line or primary cells. In contrast, small cell colonies were visible on Sp2/0 cell-plated agar plate as early as day 6, which became much larger by day 10 after plating (Fig. 8).

Fig. 8
figure 8

Clone formation of PFC cell line. Primary PFCs (a), PFC cell line (b) or mouse myeloma cell line Sp2/0 (c) were plated on 0.7 % soft agar plates and cell colonies were observed under light microscope (×25) after incubation for 2 weeks

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Discussion

The main methods for producing genetically engineered cells use viral vectors, of which safety issues and manufacturing costs remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture times (Kacherovsky et al. 2012). Furthermore, the transcriptional activities of viral promoters are prone to inactivation few weeks after modification (Qin et al. 2010; Scharfmann et al. 1991), which is detrimental for generating cell lines. In recent years, the SB transposon has been emerging as the favorable gene delivery system because of its random integration profile and the lack of similar transposon-like elements in animal genomes, which significantly minimizes the risk often presented by viral-based methods (Kolacsek et al. 2011). Compared to the previous methods for generating pig cell lines, the new method established in this study had the following improvements. First, for the first time pTERT cDNA was used as the immortalization gene of pig cells which should express a more authentic telomerase enzyme than hTERT cDNA. Second, the pEF-1α promoter was used to drive ectopic pTERT expression and the stable expression was maintained after repeated passages. Finally, by combining the SB transposon system with dilution cell cloning, the PFC cell line generated had no antibiotic resistance gene or viral sequence which minimized the safety concern associated with plasmid- or viral-based methods.

Our pilot experiment confirmed that the SB transposon system is a highly efficient gene delivery vehicle (Izsvák et al. 2009). After passages for 7 generations, for example, much more GFP-positive cells remained in the PFC culture transfected with the transposon system than for those transfected with the transposon vector alone (Fig. 2). The same conditions were used to transfect primary PFCs with the SB transposon system. Among ten cell clones identified, however, only two contained a pTERT expression cassette and the remaining contained randomly integrated intact vector. This could be due to the use of early time for cell cloning (after 1 passage) at which time residual free pTEG-TERT vector may be still present in some cells. Although the exact reason remains to be defined, it is worth to consideration for generation of animal cell lines without an antibiotic resistant gene and/or viral sequence.

Both RT-PCR and ELISA assays showed that the pTERT cDNA cloned from pig kidney cell line encoded a functional telomerase enzyme (Fig. 4a). Furthermore, the ectopic expression of pTERT in PFC cell line was stable during different passages, indicating the pEF-1α promoter was not prone to inactivation by repeated passage. It should be noted that the endogenous telomerase activity was also expressed in primary PFCs before passage 5 (Fig. 4b). This may be the reason for active proliferation of primary PFCs before passage 5 and reaching senescence by passage 10. The expression of endogenous telomerase in primary pig cells has been reported previously (Gomes et al. 2011).

Our cell characterization studies showed that the pTERT-immortalized PFC cell line kept the growth properties of primary cells, including contact growth inhibition in FBS-containing medium, population doubling time and cloning efficiency (Table 2). The cell cycle analysis showed the cell cycle distribution of PFC cell line was similar to the primary cells with a limited number of apoptotic cells detected (Fig. 7). Furthermore, the growth of PFC cell line was anchorage-dependent (Fig. 8), indicating the lack of anchorage-independent growth which is one of the hallmarks of cell transformation (Akagi et al. 2003).

In some cases, hTERT alone is sufficient for immortalization of some cell types such as foreskin fibroblasts (Bodnar et al. 1998) and endothelial cells (Chang et al. 2005). However, this is not the case for other cell types such as human epithelial cells (Kiyono et al. 1998). Even fibroblasts derived from lung can not be immortalized with hTERT alone (Itahana et al. 2003), and additional inactivation of the p16INK4a/Rb pathway is required for efficient immortalization (Kiyono et al. 1998; Dickson et al. 2000; Taylor et al. 2004). More recent studies have also shown that the efficient immortalization of human cells requires the combination of p16INK4a or p53 suppression with hTERT overexpression (Haga et al. 2007; Liu et al. 2013). However, contradictory evidence has also be found in other studies. For example, it has been shown that p16INK4a inactivation is not required to immortalize human mammary epithelial cells. The premature growth arrest and loss of p16 in several epithelial cell types are due to inadequate growth conditions that can be overcome by the use of feeder layers (Herbert et al. 2002). A more recent study has also shown that hTERT alone can immortalize epithelial cells of renal proximal tubules without changing their functional characteristics (Wieser et al. 2008). Therefore, whether hTERT alone is sufficient to immortalize human cells is still a matter of controversy, which may be cell type- and/or culture condition-dependent (Liu et al. 2013). In this study, we characterized only one PFC cell line, which was just a tiny fraction of the number of transfected cells. Further studies are needed to clarify whether the cell line was immortalized by pTERT alone or by a second event such as loss of p16 function.

Conclusions

The new method established herein is useful for generating pig cell lines without an antibiotic resistant gene and viral sequence. The PFC cell line generated may be more suitable for pig physiological analyses and/or vaccine development.