Pseudo attP sites in favor of transgene integration and expression in cultured porcine cells identified by streptomyces phage phiC31 integrase
© Bi et al.; licensee BioMed Central Ltd. 2013
Received: 16 April 2013
Accepted: 27 August 2013
Published: 8 September 2013
Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5′ and 3′ junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the position effect of identical transgene located in diverse chromosomal contexts. These findings also form the basis for targeted pig genome engineering and may be used to produce genetically modified pigs for agricultural and biomedical uses.
KeywordsPhiC31 integrase Pig Pseudo attP site TAIL-PCR
Transgene technology holds promising applications in biomedicine and agriculture. Classical DNA pronuclear microinjection is a reliable tool to produce transgenic animals, but the inefficiency and uncontrollability of integration site and copy number are the major limitations . Retrovirus and transposons enhance gene transfer efficiency and achieve single-copy transgenesis, but these methods integrate transgene throughout the genome [2–6]. As a result, the transgene is likely to disrupt endogenous gene structure. Also, it is still subjected to position effect that results in expression variation, even transgene silencing .
These problems can be overcome by targeting the transgene to a specific genomic site via homologous recombination (HR). However, it is usually labor-intensive and time-consuming due to the extremely low frequency of HR in mammalian cells . Recently developed hybrid nucleases like ZFN, TALEn and Cas9 were believed to have a fair targeting efficiency, but it is still challenging to screen nucleases with high affinity and specificity [9, 10]. Hence, molecular tools that introduce site-specific transgene integration with robust gene expression are necessary for precise transgenesis applications.
Phage integrases carry out irreversible and unidirectional recombination between attachment sites of phage and bacteria genomes, known as attB and attP sites, respectively . Interestingly, these prokaryote-derived integrases also function in eukaryotic cells and has been used to target transgene to specific sites in fly, silkworm, mouse and rat [12–15]. These native docking sites in eukaryote is called pseudo attP site. PhiC31 integrase system has become a powerful tool to produce transgenic animals . However, few studies have explored its potential to modify livestock genomes, except that specific pseudo attP sites have been identified in bovine genome [17–19]. In particular, as pig is an economically important and biomedically significant model animal, efficiently targeted genome engineering method is a necessity. In this study, we demonstrated usefulness of PhiC31 integrase to mediate site-specific transgene integration into pig pseudo attP sites. We also performed a functional rescue assay to reconstitute the recombination activity of pig pseudo attP sites. Our data indicate that pig pseudo attP sites are in favor of transgene expression. This work paves a new way to conduct targeted pig genome engineering.
An optimal molar ratio of PhiC31 integrase to substrate is important to its activity in pig cell environment
To know if PhiC31 integrase system functions in pig cell environment, we used pCMV-Int and pBCPB+ plasmids to detect extra chromosomal intramolecular recombination. The two plasmids contain the required components of PhiC31 system: the integrase, attB and attP recognition substrates placed in direct orientation. In this context, PhiC31 integrase will catalyze the recombination between attP and attB site, deleting LacZ gene and yielding attL and attR hybrid sites. Forty-eight hours post transfection of the two plasmids into pig kidney PK15 cell line, a cell PCR assay was conducted to detect attL and attR sites with specific primers. Expected products were only obtained from the co-transfection of both the two plasmids. Neither pCMV-Int nor pBCPB+ produced these amplicons. We also titrated the molar ratio between enzyme and substrate. It showed that molar ratio of 5:1 (pCMV-Int: pBCPB+ = 5:1) resulted in maximum recombination efficiency 82%. The result suggested that the three components of PhiC31 integrase system were sufficient to induce site-specific intramolecular recombination in pig cells, implying that no cofactors were required for PhiC31 integrase to function in pig cells.
Identification of candidate pig pseudo attP sites
Pseudo attP sites in pig kidney PK15 cell line
Identity to attP (%)c
AAGGA TTTGG ATT TCATC TGT GTAT GCTCAGTACTTTTT
Chr1, † , 114220089–114220127,Int.Ge
TGGATTTGGG TG CCAC GG TGA CTCAGATGGGAT GCCGGG
Chr7, − , 45581124–45581162,Int.G
2/6 = 33%
GCC TGGTATCATCAA ACA TTT A TGGGATCTCTGCCATG T
Chr9, − , 42746366–42746404,Int.G
1/6 = 17%
ATAC CCC CAATATA GC TC TGA A AT AC CAATGG GTG TTCC
Chr3, - , 54698718–54698756,Int.G
1/6 = 17%
Sequencing of the TAIL-PCR products further revealed the covalent linkage of reporter plasmid in PK15 cellular genome. The joining sites were in accordance with PhiC31 integrase-mediated attB and attP crossover reaction. It implies that PhiC31 integrase functions in pig cells to introduce attB-containing plasmid into pseudo attP sites. Meanwhile, we observed imperfect repairing in the junction sites (Figure 3B). For example, in the 5156 pseudo attP site, the recombination brought a micro insertion of 4 nucleotides between PK15 cellular genome and attR site. Micro deletion of 2 nucleotides occurred at the recombination site of 2015 pseudo attP site (Figure 3C). In Figure 3B, it was shown that micro mutations could occurr at attL or attR sites. We did not find large mutation as reported previously .
Functional rescue assay proved that candidate pig pseudo attP sites were bona fide recombination sites
Pig pseudo attP sites are in favor of robust transgene expression
In this paper we demonstrated the catalyzing activity of PhiC31 integrase to introduce site-specific transgene integration into PK15 cellular genome. We showed that PhiC31 integrase system work synergistically in a quantitative manner. For the extra chromosomal site-specific recombination, it was found that the optimal ratio of pCMV-Int (6230bp) to pBCPB+ (7221bp) is 5:1 in pig cellular environment. PhiC31 integrase catalyzes the recombination as high as 82%. In an attempt to examine the potency of PhiC31 integrase-mediated intermolecular recombination, we also observed that a 5:1 molar ratio of pCMV-Int to pEGFP-N1-attB resulted in robust and long term EGFP expression and produced maximum percentage of EGFP-positive cell clones. Please note that in all the transfections we have assured the charge ratio of DNA plasmid to transfection reagent invariable. Hence, our results allow for a direct comparison among diverse transfection settings. Such results were also observed in previous reports [17, 20]. Exact explanations of the “overproduction-inhibition effect” remain unknown. But careful examination of molar ratio of enzyme to substrate is a critical step prior to its successful use. This also suggests that, given that an unsuitable enzyme/substrate ratio is used, the efficacy of this system will be depressed.
Secondly, as documented previously in human, mouse, rat, fruit fly, silkworm and cattle, pseudo attP site profile varies across species . Four pseudo attP sites were mapped in PK15 cellular genome in this work. All of them were located in intergenic regions, and none was found in other mammalian genomes. This indicates that PhiC31 integrase-mediated transgene integration pattern is species-specific, and the sites identified in this work are unique to pig. Our findings also suggest that pseudo attP sites in mammalian genome are not conserved during evolution. Additionally, this is unlike the integration pattern from virus gene transfer system, such as MLV that shows a strong preference for transcription start sequences . In this regard, PhiC31 integrase functions in a safe way to modify host genome. On one hand, PhiC31 integrase is not likely to induce endogenous gene mutagenesis in pig cells. On the other hand, we did not observe any aberrant morphology and abnormal proliferation in the transgenic cell lines. Thus they are in favor of transgene integration and expression. Especially, 5113 and 5156 pseudo attP sites are ideal locus for robust transgene expression. This is particularly advantageous to gene therapy and safe transgenic technology, as this will reduce the risk of insertional mutagenesis [25, 26].
Thirdly, we reconstituted the site-specific recombination by performing a functional rescue assay. To our knowledge, this is the first report to prove candidate pseudo attP site to be bona fide attP site. Through this test, we ruled out the possibility that pEGFP-N1-attB happened to be randomly integrated into candidate pig pseudo attP sites. This also indicates that the recognition site of PhiC31 integrase in PK15 cellular genome is not stringent, as the maximum similarity to wild type attP is around 30%. We observed an inverse correlation between similarity and recombination efficiency. It implies that there might be certain endogenous factors affect the sequence specificity of PhiC31 integrase . But this needs a further research of binding proteins interacting with PhiC31 integrase. Hence, improvement of its recognition specificity is necessary in future studies to prevent aberrant recombination and multiple site integration .
Finally, we carried out TAIL-PCR to isolate the integration site. In previous studies, inverse PCR, half-nested PCR and plasmid rescue were often used to identify the candidate pseudo attP sites. But the common drawback of these methods is that they require a large starting amount of genomic DNA (usually 10 μg) for restriction and ligation. However, TAIL-PCR needs as little as 0.5 μg genomic DNA. In this work, we obtained four pig pseudo attP sites by TAIL-PCR. Moreover, the integration of reporter plasmid into all these four pseudo attP sites could be proved by junction PCR, indicating that the positive rate of TAIL-PCR is 100%. Accordingly, we believe that TAIL-PCR is a valuable tool owing to its ease, low cost and high reproducibility.
Our data demonstrated that PhiC31 integrase functions efficiently for site-specific transgene integration into PK15 cellular genome. We isolated four pig pseudo attP sites and proved their recombination activity by a functional rescue assay. PhiC31 integrase induces mono-allelic transgene integration in pig cells. Pig pseudo attP sites were proved to be in favor of robust transgene expression. Our findings also established an ideal model to study the position effect of identical transgene structure located in diverse chromosomal contexts. These results form the basis for targeted pig genome engineering and may be used to produce genetically modified pigs for agricultural and biomedical uses.
Plasmid construction and DNA manipulation
The plasmid pCMV–Int (expressing PhiC31 integrase) and reporter plasmid pBCPB+ were gifts from M. P. Calos (Stanford University, USA). A Roche high pure PCR template preparation kit was used to extract genomic DNA. All plasmids were prepared by a TIANpure midi plasmid kit (Tiangen, Beijing, China). A Nanophotometer P-class was used to check the quality and quantity of DNA (Implen, Germany). AttB fragment was amplified from pBCPB+ and inserted into the AseI site of pEGFP-N1 (Clontech), and the resultant plasmid was pEGFP-N1-attB. For ABI-REC to create p’BCPB+ plasmids, pig pseudo attP sites were amplified by a KOD plus high-fidelity DNA polymerase and fused into pBCPB+ plasmid, replacing the wild-type attP site. For details of ABI-REC protocol, please refer to previous description . Briefly, ABI-REC is a restriction-free DNA cloning method owning to asymmetric 3-primer PCR and intramolecular homologous recombination in bacteria. For extra chromosomal DNA recombination, we used a PCR assay with cell lysis to detect the hybrid sites. Briefly, 103 cells were disrupted in lysis buffer (0.005% SDS and 1 mg/ml proteinase K) and incubated at 55°C for 1 hour and 94°C for 5 minutes. The lysis solution was centrifuged at 12000 rpm for 1 minute and the supernatant was transferred into a fresh tube. The supernatant contained chromosomal and extra chromosomal DNA and was used as template in a one-stop PCR . Sequences of p’BCPB+ plasmids were supplemented in additional files. BglII, PstI, NdeI restriction nucleases (Fermentas, Lithuania) were used to checked the presence of pig pseudo attP sites.
Cell culture, transfection and stable cell line selection
Pig kidney PK15 cell line was cultured in DMEM (Dulbecco’s modified Eagle’s medium, Gibco, Life Technologies) supplemented with 10% (v/v) FCS (fetal calf serum, Hyclone), 100 IU/ml penicillin and 0.1 mg/ml streptomycin. Cells were grown at 39°C in the presence of 5% CO2 and were subcultured (1:5) every 3 days. To obtain stable transfected cells, PK15 cells were split in a 35-mm plate until they reached 80% confluency. Cells were washed with PBS and then incubated with a mixture of 4 μg of DNA, 10 μl of Lipofectamine 2000 (Invitrogen) and 500 μl of Opti-MEM that was prepared as described by the manufacturer. To ensure the constant charge ratio of total nucleic acid and lipid, “filler or unrelated DNA” (pUC19 plasmid) was added to the transfection mixture. Following 6 h incubation, the medium was replaced with fresh medium. 48h post transfection, 600 μg/ml G418 (Sigma) was added. After 2 weeks of selection, G418-resistant colonies were picked manually and maintained in media with 50 μg/ml of G418. For cell proliferation assay, an Alamar blue kit (Gibco) was used. Briefly, 1000 cells were seeded in a 96-well plate in triplicate. 10 μl Alamar blue indicator was added into the medium (with a final concentration of 10% v/v). The plate was incubated for additional 5 hours. The optical densities were measured at 570 nm and 630 nm in a micro-plate reader (Multiskan MS; Labsystems, Helsinki, Finland). Reduction percentage was calculated according to the formula provided in the instruction (Gibco). Fresh medium was used as blank control. Such a procedure was repeatedly performed for continuous 15 days. The reduction values at day1, day5, day10 and day 15 were shown in the manuscript (Figure 5C).
Extracellular EGFP quantification
Extracellular EGFP into medium was quantified using a Glomax Multi Jr fluorescence detector, according to the manufacture’s manual book. The EGFP level was indicated in arbitrary units. In order to normalize the input variation, an ELISA assay was used to measure the IGF-I expression level secreted into the medium as an internal control. Briefly, a 96-well plate was coated with anti-pig IGF-I antibody (Abcam, Cambridge, UK). After blocking, extracellular proteins were added in triplicate. IGF-I expression was then determined using a horseradish peroxidase-conjugated secondary antibody (Abcam) with 3, 3′, 5, 5′-tetramethylbenzidine (Sigma-Aldrich) as the substrate. The reaction was stopped by addition of 100 μL of 1 M H2SO4. Absorbance was measured at 450 nm using an ELISA reader (Bio-TEK Instruments Inc, Winooski, VT, USA).
The cells were washed thrice with ice-cold PBS to remove residual medium as much as possible. Afterwards the cells were lysed by NP40 Cell Lysis Buffer (Invitrogen) with phenylmethylsulfonyl fluoride (PMSF). After centrifugation, the supernatants were collected and quantified by Pierce BCA protein assay kit (Thermo Scientific). The total proteins were denatured by boiling for 5 minutes in a water bath and chilled in ice. 15% SDS-PAGE was used to size-fractionate the samples. Then they were transferred electrophoretically onto polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ). After blocking with 2% milk, the membranes were incubated with primary antibodies at 4°C for 1 hour. The monoclonal primary antibody against EGFP (1:1000) was purchased from ProteinTech Co. Ltd. (Wuhan, China). The monoclonal primary antibody against β-actin (1:10000) was purchased from Sigma. After washing with PBST, the membranes were incubated with rabbit anti-mouse horseradish peroxidase-conjugated secondary antibodies (Sigma) for 2 h at room temperature. The signals were visualized by enhanced chemiluminescence (Perkin-Elmer, Norwalk, CT).
Primers used in this study
To amplify 400 bp attB site in AseI of pEGFP-N1
To detect the attL site, 400 bp
To detect the attR site,520 bp
TAIL-PCR to identify integration site
Use with IV-R1 for determination of 5′ end of 5113 pseudo attP site
Use with IV-F2 for determination of 3′ end of 5113 pseudo attP site
Use with IV-R1 for determination of 5′ end of 5156 pseudo attP site
Use with IV-F2 for determination of 3′ end of 5156 pseudo attP site
Use with IV-R1 for determination of 5′ end of 1015 pseudo attP site
Use with IV-F2 for determination of 3′ end of 1015 pseudo attP site
Use with IV-R1 for determination of 5′ end of 2015 pseudo attP site
Use with IV-F2 for determination of 3′ end of 2015 pseudo attP site
ABI-REC cloning of 5113 pseudo attP site in pBCPB+ plasmid
ABI-REC cloning of 5156 pseudo attP site in pBCPB+ plasmid
ABI-REC cloning of 1015 pseudo attP site in pBCPB+ plasmid
ABI-REC cloning of 2015 pseudo attP site in pBCPB+ plasmid
ABI-REC cloning of pig pseudo site
Real-time PCR primers, 130 bp
Real-time PCR primers, 81 bp
A Toyobo real-time SYBR Green I PCR master mix was used to amplify EGFP or TFRC gene in a Rotor Gene 6000 real-time rotary analyzer (Corbette Lifescience, Australia). The 20 μl PCR reaction mixture contained 10 μl 2 × master mix, 0.3 μl primer mix (5 μM each), 0.5 μl genomic DNA (50 ng) and 9.2 μl PCR-grade water. A two-step amplification protocol was used with the following parameters: 95°C for 2 minutes to pre-denature the template and activate Taq DNA polymerase followed by 40 cycles each of denaturation at 95°C for 8 seconds, annealing and extension at 60°C for 25 seconds. A final melting temperature analysis from 50°C to 99°C was used to ensure amplicon uniformity. Fluorescence was acquired at the step of annealing and extension. All PCR amplifications were performed in triplicate for each treatment. Wild-type PK15 genomic DNA was used as negative control. EGFP standard curve was produced by amplifying pEGFP-N1-attB at gradient concentration of 10 ng, 1 ng, 100 pg, 10 pg, 1 pg. TFRC standard curve was produced by amplifying wild-type gDNA at gradient concentration of 100 ng, 10 ng, 1 ng, 100 pg, 10 pg. Primers used for real-time PCR (Table 2) were designed and selected by Primer Premier 5. Relative signal intensities were calculated by the built-in software RG6000 series 1.7. Copy number of EGFP gene was calculated with the following formula: transgene copy number/pig genome = molecules of EGFP/molecules of TFRC × 2. TFRC is a single-copy gene in haploid pig genome.
Statistical and bioinformatic analysis
The results of colony calculation, real-time PCR, EGFP expression were shown as the mean ± SD (n = 3). The statistical analysis was performed using Student’s t-test for comparison between two groups. The difference was considered significant if p < 0.05. PCR product quantification is carried out by VisionCapture . The identity of pig pseudo attP site to wild-type attP site was determined using Bioedit.
Thermal asymmetric interlaced PCR
Asymmetric bridge PCR and intramolecular homologous recombination
Enhanced green fluorescence protein
Attachment site of bacteria
Attachment site of phage
Left attachment site
Right attachment site
Enzyme-linked immunosorbent assay
Insulin-like growth factor I
The authors are grateful to M. P. Calos (Stanford University, USA) for her generosity to provide us expression plasmids. We are also grateful to Dr. Qingwen Ma (Shanghai Jiaotong University) and Dr. Ruhong Jiang (Applied Stem Cells, Inc., USA) for their helpful discussions. We would like to thank the assistance of team of veterinary medicine from Hubei Academy of Agricultural Science in ELISA assay. This study was supported by Natural Science Foundation of China (31201790), International Science and Technology Cooperation Project of Hubei Province (2009BFA012), a Key Project of Youth Foundation (2011NKYJJ13) of Hubei Academy of Agricultural Science (HAAS), Innovation Center for Agricultural Sciences and Technologies of Hubei Province (2011-620-001-003), open grant from Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding (2013ZD120/121). It was also supported by China Major Program of Genetically Modified Organism New Species Cultivation (2013ZX08010-003, 2013ZX08006-003, and 2013ZX08006-002).
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