Stem Cells
Vol. 25 No.
4
April 2007, pp.
1055
-1061
doi:10.1634/stemcells.2006-0616; www.StemCells.com
© 2007 AlphaMed Press
Baculoviral Vector-Mediated Transient and Stable Transgene Expression in Human Embryonic Stem Cells
Jieming Zenga,
Juan Dua,b,
Ying Zhaoa,b,
Nallasivam Palanisamyc,
Shu Wanga,b
aInstitute of Bioengineering and Nanotechnology, Singapore;
bDepartment of Biological Sciences, National University of Singapore, Singapore;
cGenome Institute of Singapore, Singapore
Key Words. Human embryonic stem cells • Baculovirus • Gene transfer • Transgene expression • Genetic modification
Correspondence: Shu Wang, Ph.D., Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos 04-01, Singapore 138669. Telephone: 65-6824-7105; Fax: 65-6478-9083; e-mail: swang{at}ibn.a-star.edu.sg
Received September 29, 2006;
accepted for publication December 27, 2006.
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ABSTRACT
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Human embryonic stem (hES) cells as a renewable cell source have great prospective applications in both developmental biology research and regenerative medicine. To realize these potentials, the development of effective and safe genetic manipulation methods in hES cells is an obvious demand. We report here that baculoviral vectors were able to transduce hES cells efficiently. In transient transduction experiments, a recombinant baculoviral vector equipped with a human elongation factor 1-
promoter and a woodchuck hepatitis post-transcriptional regulatory element transduced up to 80% of cells in hES cell clumps and embryoid bodies. For prolonged transgene expression, hybrid baculoviral vectors that have incorporated a rep gene and inverted terminal repeat sequences from adeno-associated virus were produced. These hybrid vectors yielded stable transgene expression during the prolonged undifferentiated proliferation of hES cells and after differentiation. Baculoviral transduction did not affect the normal growth, phenotype, and pluripotency of hES cells. Thus, baculoviral vectors suitable for both transient overexpression and long-term stable expression are an attractive option for genetic manipulation of hES cells.
Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION
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Versatile gene transfer vectors capable of mediating either transient or prolonged transgene expression are required in genetic engineering of human embryonic stem (hES) cells for the purposes of both regenerative medicine and basic biological studies. The technical development of gene transfer into hES cells has recently been reviewed [1, 2]. Human immunodeficiency virus (HIV)-1-based lentiviral vectors were the first viral vectors used to genetically engineer hES cells [1]. Lentiviral vectors produce high levels of transduction efficiency and stable transgene expression as they integrate into the host genome and are resistant to transcriptional silencing [3–5]. However, because of the severe pathogenic effects of HIV-1 replication in humans, the potential emergence of replication-competent retrovirus from HIV-1-based vectors raises concerns over their use in clinical settings. In addition, random chromosome integration of lentiviral vectors poses the risk of insertional mutagenesis, oncogene activation, and cellular transformation. The development of leukemia in two children after gene therapy for SCID-X1 [6] has brought intense scrutiny to the potential risk. In addition, lentiviral vectors may not be suitable for transient transgene expression. Viral vectors derived from adenovirus and adeno-associated virus (AAV) have a much lower risk of insertional mutagenesis and have been tested in hES cells, but their transduction efficiencies were less satisfactory [7]. Nonviral methods, such as chemical-based plasmid delivery and electroporation, have also been used for gene transfer into hES cells [8, 9]. These methods, however, suffer from low levels of transient transfection or cause membrane damage. When combined with antibiotic resistance gene and drug selection for stable clones, the nonviral methods provide a random chromosome integration of the transgene.
The insect baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-based vectors have recently been introduced as a new type of delivery vehicle for transgene expression in mammalian cells [10]. The virus can enter mammalian cells but does not replicate, and it is unable to recombine with pre-existing viral genetic materials in mammalian cells. Inside mammalian cells, baculoviral vectors produce little to no microscopically observable cytotoxicity even at a high multiplicity of infection (MOI). One significant advantage of using baculovirus AcMNPV as a gene delivery vector is the large cloning capacity to accommodate up to 30 kilobases (kb) of DNA insert, which can be used to deliver a large functional gene or multiple genes from a single vector. Although baculoviral vectors have been tested in human mesenchymal stem cells [11], their efficiency remains to be proven in the context of human embryonic stem cells. In the present study, we have examined whether baculovirus can be used to transduce hES cells.
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MATERIALS AND METHODS
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Vector Construction and Virus Preparation
To generate recombinant baculoviral vectors, the transfer plasmid pFastBac1 from Invitrogen (Carlsbad, CA, http://www.invitrogen.com) was used. For transient expression, one vector was constructed by inserting between BamHI and EcoRI of pFastBac1 a human elongation factor-1 (EF1) promoter from pEF1V5-HisA (Invitrogen). Then, the enhanced green fluorescent protein (eGFP) gene from pEGFP-C1 (Clontech, Mountain View, CA, http://www.clontech.com) was amplified by polymerase chain reaction (PCR) and inserted between EcoRI and SpeI. To incorporate the woodchuck hepatitis post-transcriptional regulatory element (WPRE) into the above vector, an eGFP-WPRE fragment was PCR-amplified from psubCMV-eGFP-WPRE (kindly provided by Professor H. Büeler, University of Zurich, Switzerland) and inserted downstream of the EF1 promoter at the EcoRI/SpeI sites to replace the eGFP gene from pEGFP-C1. In another transient expression vector, the human cytomegalovirus immediate-early gene promoter and enhancer (cytomegalovirus [CMV] promoter) and eGFP gene from pEGFP-C1 (Clontech) was inserted between BamHI and EcoRI of pFastBac1.
For stable transgene expression, recombinant pFastBac1 vectors accommodating the AAV rep 78/68 genes and inverted terminal repeat (ITR) sequences were constructed through a multiple-step cloning procedure. The construction started from inserting between the AvrII and SalI sites of a pFastBac1 vector a fragment of pAAV plasmid containing an expression cassette with a multiple cloning site, a reporter gene encoding eGFP, a simian virus 40 poly(A) signal, and two ITR sequences at both ends [12]. Then, the CMV promoter from pRc/CMV2 or the EF1 promoter from pEF1V5-HisA was inserted into the recombinant pFastBac1 between KpnI and HindIII of the pFastBac1 vector. In the third step, a 4.4-kb fragment of the AAV type 2 genome from pSub201 (kindly provided by Professor R.J. Samulski, University of North Carolina, Chapel Hill, NC) was PCR-amplified and inserted into another pFastBac1 vector at RsrII site, from which a 1.5-kb fragment that contained the encoding sequence of Cap and Cap terminator was removed by digestion with ApaI. After the remaining part of the second recombinant pFastBac1 was religated, a 3.0-kb fragment from this religated vector was cut with RsrII and then inserted into the first recombinant pFastBac1. This 3.0-kb fragment contains the full sequence of rep gene with p5, p19, p40, and Rep terminator and was placed outside the ITRs in an antisense orientation with respect to the pPolh promoter of pFastBac1 (Fig. 2A).
Recombinant baculoviruses with the above expression cassettes were produced and propagated in Sf9 insect cells according to the manual of the Bac-to-Bac Baculovirus Expression system (Invitrogen). Budded viruses in the insect cell culture medium were filtered through a 0.2-µm pore size filters (Millipore, Billerica, MA, http://www.millipore.com) to remove any cell debris, and concentrated by centrifugation at 28,000g for 60 minutes. Viral pellets were resuspended in appropriate volumes of 0.1 M phosphate-buffered saline (PBS) and their infectious titers (plaque-forming units [pfu]) were determined by plaque assay on Sf9 cells.
Maintenance and Differentiation of hES cells
The NIH Human Embryonic Stem Cell Registry-listed hES cell line HES-1 [13] and its feeder cell K4 mouse embryonic fibroblasts (mEFs) were obtained from ES Cell International (ESI; Singapore, http://www.escellinternational.com). The hES cells were amplified and maintained according to the protocol provided by ES Cell International. For viral transduction experiments, this HES-1 line was cultured on mitotically inactivated mEFs (CF-1 from American Type Culture Collection, Manassas, VA, http://www.atcc.org) seeded in gelatin-coated dishes in 80% knockout Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 20% Knockout Serum Replacement (Invitrogen), 2 mM L-glutamine (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 0.1 mM 2-mercaptoethanol (Invitrogen), 4 ng/ml basic fibroblast growth factor (bFGF) (Invitrogen), 50 U/ml penicillin, and 50 µg/ml streptomycin [14]. The hES colonies were subcultured every 7 days by mechanical slicing and replating into fresh feeder layers.
To form embryoid bodies (EBs), hES cells were grown to form large colonies and detached by using 0.1 mg/ml Dispase (Invitrogen). The hES cell clumps were transferred to a 15-ml conical tube containing 10 ml of a differentiation medium consisting of 80% knockout DMEM, 20% fetal bovine serum (Hyclone, Logan, UT, http://www.hyclone.com), 2 mM L-glutamine, and 0.1 mM nonessential amino acids and allowed to settle to the bottom. The supernatant was removed. The cell clumps were resuspended in the differentiation medium and transferred to a Petri dish. The cells were fed every day by replacing half of the medium with fresh differentiation medium and were cultured for 1 week.
The derivation of neurons from hES cells was achieved based on the method described by Reubinoff et al. [15]. In brief, the hES cell differentiation was induced by prolonged culture for 3–4 weeks on feeders. The distinct areas with uniformly white-gray and opaque appearance under dark-field stereomicroscope were cut and dissected into small-cell clumps. These cell clumps were then transferred to low cell binding six-well plates (Nalge Nunc International, Rochester, NY, http://www.nuncbrand.com) containing DMEM/Ham's F12 medium (1:1; Invitrogen) supplemented with B-27 (1:50; Invitrogen), 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 20 ng/ml human epidermal growth factor (EGF) (Chemicon, Temecula, CA, http://www.chemicon.com) and 20 ng/ml bFGF (Chemicon). After culture for 1–3 weeks, round neural spheres were formed. The spheres were then plated into dishes coated with poly(D-lysine) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and laminin (Sigma-Aldrich). Neuronal differentiation was induced by the withdrawal of the growth factors from the culture medium.
Viral Transduction and FACS Analysis to Quantify Transduction Efficiency
For viral transduction, hES cell clumps in suspension were used to eliminate the effect of virus absorption by mEFs [4]. At the time of routine passage, hES cell clumps were isolated from hES colonies by mechanical slicing. For each transduction, eight hES clumps were suspended in 50 µl of knockout DMEM in an Eppendorf tube, and baculoviral vectors in 50 µl of PBS were added at an MOI of 100. After incubation for 2 hours, the clumps were washed with PBS and replated onto fresh mEF feeder.
Fluorescence-activated cell sorting (FACS) analysis was used to quantify the transduction efficiency of baculoviral vector. After Dispase digestion to detach hES cell colonies from the mEFs, the colonies were collected, washed in PBS, and trypsinized to single cells before being analyzed with FACSCalibur flow cytometer (BD Biosciences, San Diego, http://www.bdbiosciences.com).
Reverse Transcription-PCR Analysis
Total RNA was extracted from hES cells or EBs using the RNeasy kit from QIAGEN (Valencia, CA; http://www.qiagen.com) according to the manufacturer's instructions. First-strand cDNA was synthesized using the SuperScript III First-Strand Synthesis System for reverse transcription (RT)-PCR (Invitrogen). One microliter of cDNA reaction mix was subjected to PCR amplification with primers shown in supplemental online Table 1. Reactions were subjected to 30 PCR cycles after denaturation at 94°C for 4 minutes as follows: 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds. An additional extension step of 72°C for 5 minutes was included at the end of the 30th cycle. PCR products were electrophoresed on a 2% agarose gel.
Immunohistochemistry and Karyotypic Analysis
For hES cell immunostaining, the colonies were washed with PBS and fixed with ice-cold absolute methanol for 15 minutes. This was followed by permeabilization with PBS containing 0.1% Triton X-100 for 30 seconds and blocking with 1% bovine serum albumin in PBS for 30 minutes. Primary monoclonal antibodies against hES cell marker TRA-1-60 (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com) or TRA-1-81 (Santa Cruz Biotechnology) were used to incubate with the samples for 1 hour. After washing, the secondary antibodies goat anti-mouse IgM-PE (Santa Cruz Biotechnology) were used for localization. To detect eGFP protein, Alexa Fluor 488-labeled rabbit anti-GFP antibody (Invitrogen) was included for double immunostaining. The samples were counterstained with Hoechst stain before observation. Likewise, the hES cell-derived neurons were analyzed by indirect immunostaining using the primary rabbit antibody against 200-kDa neurofilament (NF200) (Sigma) and the secondary swine anti-rabbit Ig/tetramethylrhodamine B isothiocyanate antibody (DAKO, Glostrup, Denmark, http://www.dako.com).
To analyze the karyotype of hES cells, cells were treated with Colcemid (Invitrogen) and subjected to standard hypotonic treatment and methanol/acetic acid (3:1) fixation. Slides were prepared by standard air-drying method and hybridized with human spectral karyotyping paint probe (Applied Spectral Imaging Inc., Vista, CA; http://www.spectral-imaging.com).
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RESULTS
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Baculoviral Vectors Effectively Mediated Gene Transfer to hES Cells
We isolated hES cell clumps of the HES-1 line [13] by mechanical slicing, suspended them in serum-free hES cell culture medium, and transduced the clumps with baculoviral vectors containing a eGFP gene at an MOI of 100 pfu for 2 hours before replating on feeders. Using a baculoviral vector incorporating the human EF1 promoter, we observed eGFP expression as early as 6 hours after transduction. The expression became intense at 24 hours, with bright green fluorescence observed in the whole clumps (Fig. 1A). hES cells in the clumps kept outgrowing after transduction, resulting in colonies with a central "green" region surrounded by newly generated, eGFP-negative hES cells (Fig. 1A, day 4). This finding suggests that the virus was unable to replicate and to deliver its genome to hES cell progeny. The eGFP signals in the central region decreased over time (Fig. 1A, days 1–6) and became very weak after day 6. This baculoviral vector-mediated transient transgene expression was reproduced in hES cells with CMV promoter-driven eGFP gene, in which the eGFP gene was expressed with a similar time course but lower intensity (see supplemental online Fig. 1). Using baculoviral vectors with both the EF1 promoter and WPRE, up to 80% of the cells in the infected hES cell clumps were eGFP-positive at day 2 as analyzed by flow cytometry (Fig. 1B); while using baculoviral vectors with the CMV promoter or the human EF1 promoter without the WPRE, the transduction efficiency was approximately 25%–40% (Fig. 1B). The baculoviral vectors were also able to transduce embryoid bodies derived from hES cells (Fig. 1C). We concluded that baculoviral vectors were effective in transducing not only proliferating undifferentiated hES cells but also the cells at an early stage of differentiation.
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Figure 1. Transient enhanced green fluorescent protein expression in human embryonic stem cells mediated by baculoviral vectors. (A): A time-course observation shows one hES cell colony 1, 2, 4, and 6 days after transduction with a recombinant baculovirus harboring the elongation factor-1 promoter-driven eGFP gene at a multiplicity of infection (MOI) of 100 pfu. Phase-contrast (left), fluorescence (middle), and merged (right) images are shown. (B): Flow cytometric analysis of eGFP-positive hES cells 2 days after baculoviral transduction at an MOI of 100 pfu. From top to bottom, recombinant baculoviral vectors accommodating the cytomegalovirus promoter, the EF1 promoter, and the EF1 promoter plus woodchuck hepatitis post-transcriptional regulatory element were used, respectively. The percentages of eGFP-positive cells are indicated. (C): An embryoid body transduced with the recombinant baculovirus with the CMV promoter at an MOI of 20 pfu. Phase-contrast (left) and fluorescence (right) images depict eGFP expression on day 1 after transduction. Abbreviations: CMV, cytomegalovirus; EF1, elongation factor-1; eGFP, enhanced green fluorescent protein; hES, human embryonic stem; SSC, side scatter; WPRE, woodchuck hepatitis post-transcriptional regulatory element.
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Hybrid Baculoviral Vectors Were Able to Provide Long-Term Stable Transgene Expression in hES Cells
Stable transgene expression is crucial for applications of hES cells in both fundamental study of early human development and in regenerative medicine. To extend the duration of baculoviral vector-mediated transgene expression, we constructed hybrid baculoviral vectors (Fig. 2A) by including the rep 78/68 genes and ITR sequences from AAV [16]. The large gene capacity of baculovirus vector allows the inclusion of these large elements into a single vector together with a transgene. These chimeric virus constructs take advantage of Rep-mediated site-specific integration into the AAVS1 site in human chromosome 19q13.3-qter by nonhomologous recombination [17]. hES cells transduced with a hybrid baculoviral vector accommodating the EF1 promoter displayed weak eGFP expression for the first 3 days. By day 7, a significant portion of the cells had become eGFP-positive. The number of eGFP-positive hES cells in the colonies was increased by mechanical selection at the time for normal subculture, and after five rounds of selection, almost the entire hES colony was eGFP-positive (Fig. 2B). This finding demonstrates that with the relatively high transduction efficiency of baculoviral vectors in hES cells and the large cloning capacity of the virus (i.e., to be able to accommodate the AAV Rep/ITR system), it has become possible to use the expression of a fluorescent protein to enrich the transduced hES cells through color selection without the use of antibiotics. The selected eGFP-positive hES cells continued to express the transgene over at least 20 passages without the transgene's being silenced (Fig. 2C), and almost all the cells in the hES cell colonies were eGFP-positive as analyzed by flow cytometry (Fig. 2D), suggesting chromosome integration of the transgene mediated by the hybrid vector. To investigate whether the integration occurred at AAVS1 site, a nested PCR was carried out to amplify the transgene-AAVS1 junctions (see supplemental online data and supplemental online Fig. 2). The DNA smears resulting from PCR fragments of various lengths indicate the variability of integration sites within the 4-kb AAVS1 region.
Differentiated Progenies of Stably Transduced hES Cells Maintained Transgene Expression
To further examine the stability of transgene expression, we investigated whether eGFP expression could be preserved after the differentiation of these stably transduced hES cells into neurons, one type of the representative cells from ectoderm, using an established protocol [15]. We constructed a hybrid baculoviral vector accommodating the constitutively active CMV promoter (Fig. 2A) to facilitate the visualization of eGFP expression in both undifferentiated and differentiated cells. hES cells stably transduced with this hybrid virus were overgrown on feeders for 4 weeks (Fig. 3A), and neural spheres were then generated from the overgrown cells by incubation in suspension in the presence of bFGF and EGF for 1–3 weeks. The resulting neural spheres retained intense eGFP expression (Fig. 3B). After the neural spheres were cultured on poly(D-lysine)/laminin-coated dishes in the absence of the growth factors, they began to display typical neural differentiation phenotype with extending neurites, yet retained the bright eGFP fluorescence (Fig. 3C, 3D). The transgene expressed in these differentiated cells for at least 50 days (the end of the experiment). Cells generated from the neural differentiation were stained positively with antibodies against neuronal marker and against GFP (Fig. 3E). Besides the neural cells, eGFP-positive beating cardiomyocytes, a representative type of cells from mesoderm, were also regularly observed (see supplemental online video 1).
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Figure 3. Maintenance of transgene expression after neural differentiation of human embryonic stem (hES) cells stably transduced with a hybrid baculovirus harboring a cytomegalovirus promoter-driven enhanced green fluorescent protein (eGFP) gene. Fluorescence images show an eGFP-positive colony overgrown on feeders for 4 weeks without subculture (A), green neural spheres generated from the overgrown hES cells (B), eGFP-positive cells with neuronal morphology within a neural sphere after plating for 2 weeks and outgrowth of eGFP-positive neurites from the neural sphere (C). (D): Neuron-like cells that have migrated out from neural spheres display eGFP in both their cell bodies and neurites. (E): Immunostaining shows the co-localization of neuronal marker 200-kDa neurofilament (red) and eGFP (green) in cells derived from the stably transduced hES cells. Abbreviation: GFP, green fluorescent protein.
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Baculoviral Transduction Had No Effect on hES Cell Growth, Phenotype, and Pluripotency
Baculovirus infection caused no microscopically observable cytotoxic effects on hES cells, even at an MOI of 1,000 pfu. The infected hES cells displayed no morphological changes or proliferation defects compared with nontransduced control hES cells (Fig. 1A). RT-PCR analysis demonstrated that the baculovirus-transduced hES cells remained positive for Oct-4 and nanog, two important markers for undifferentiated hES cells, but negative for representative markers for the three germ layers such as Pax6, Neuro-D, Brachyury T, and -fetoprotein (Fig. 4B) as their nontransduced counterparts (Fig. 4A). Immunostaining showed the expression of TRA-1-60 and TRA-1-81 (Fig. 4C), further confirming the undifferentiated state of these infected hES cells. To determine the effect of baculoviral transduction on the hES cell pluripotency, the infected hES cells were used to form embryoid bodies. RT-PCR analysis showed that these embryoid bodies still expressed markers for the three germ layers (Fig. 4D) as those generated from the mock-infected hES cells (Fig. 4E), suggesting that the baculovirus-infected hES cells remained pluripotent. We also confirmed that the stably transduced hES cells grew normally as flat colonies resembling their nontransduced hES cells (Figs. 2C, 5A), expressed markers for embryonic stem cells (Fig. 5A), and retained a normal karyotype (Fig. 5B). These findings, plus the above observations that stably transduced hES cells were able to differentiate into neurons and cardiomyocytes, indicate that baculoviral infection did not affect either the self-renewal capacity or pluripotency of hES cells.
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Figure 4. Effects of baculovirus infection using transient expression vectors on the expression of molecular markers for human embryonic stem (hES) cells and the three germ layers in embryoid bodies. The hES cell clumps were transduced with recombinant baculovirus containing the enhanced green fluorescent protein gene under control of the cytomegalovirus promoter at a multiplicity of infection of 100 plaque-forming units and replated onto fresh mouse embryonic fibroblast feeders. Seven days after transduction, reverse transcription-polymerase chain reaction (RT-PCR) (A, B) and immunostaining (C) were carried out to detect the expression of molecular markers in mock-transduced hES cells (A) and hES cells transduced with baculoviral vectors (B, C). Mock-transduced (D) and baculovirus-transduced (E) hES cells were used to generate embryoid bodies and, 7 days after embryoid body formation, RT-PCR was carried out to detect the expression of markers for three germ layers in embryoid bodies. Abbreviation: AFP, -fetoprotein.
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Figure 5. Effects of baculovirus infection using stable expression vectors on the expression of molecular markers and the karyotype of stably transduced human embryonic stem (hES) cells. (A): Images of a transduced hES cell colony with stable enhanced green fluorescent protein expression collected at 15 weeks of continuous passage. (B): Spectral karyotyping analysis of stably transduced hES cells after 10 weeks of continuous passage demonstrates that the cells are karyotypically normal. A total of 20 metaphase cells were analyzed. The chromosome pattern is 46, XX. No numerical or structural aberration was observed. Abbreviation: eGFP, enhanced green fluorescent protein.
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DISCUSSION
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Baculovirus can function as a versatile vector for transient transgene expression in many types of mammalian cells [10]. The current study has expanded its use to transient and stable transduction in hES cells and their derivatives. Effective transient gene expression from episomal baculoviral vectors in hES cells might be particularly suitable for switching on or off the expression of certain "master regulatory genes" that control differentiation within a narrow time window [7, 18]. Moreover, the high-level transduction, low cytotoxicity, relatively easy construction of vectors, and efficient production of high-titer virus make baculoviral vectors a useful tool for the rapid screening of various genes to gain insight into the complex genetic networks underlying the differentiation of hES cells.
This study has further highlighted the advantage of the large cloning capacity of baculovirus, which allows the incorporation of several additional functional and regulatory elements, such as the Rep/ITR chromosome integration system of AAV, into one single vector, in addition to accommodating an expression cassette. Many studies have confirmed that AAV and hybrid viral systems that include the rep gene and ITRs are preferentially integrated at the AAVS1 site in human chromosome 19 [17]. Such chromosome integration at a defined genetic locus is preferable to random integration for the purpose of stable transgene expression. The 4-kb AAVS1 site constitutes at least part of a transcription unit [19]. It is still unclear whether disruption of the AAVS1 by Rep/ITR-mediated integration has any side effects. Nevertheless, the effects of the integration at the AAVS1 site on cell functions can be determined empirically, providing potential advantages over random integration with its unpredictable consequences.
The chromosome integration rate of a gene delivery system is one of the important issues for genetic modification of hES cells. In one early study, plasmids containing drug-resistant gene were used to transfect hES cells via Exgen, a chemical-based gene delivery method [8]. Under drug selection pressure for 14 days, stable clones can be produced at a rate of 1/100,000. However, no long-term study was conducted to investigate the maintenance of transgene expression during hES cell self-renewal or after their differentiation. Lentiviral vector is currently popular for stable genetic engineering of hES cells. The lentivirus-mediated transduction could result in transgene expression up to 48% on day 7, and after mechanical selective weekly passage of regions of hES colonies that are eGFP-positive for 7 weeks, 80% of the hES cells expressed the transgene [4]. Obviously, effective chromosome integration is one of the major advantages of lentivirus vectors in establishing stable cell lines. In our hybrid vector systems, the AAV rep gene driven by AAVp5 promoter was placed into the same baculoviral vector backbone together with the transgene. With these systems, a unique transgene expression profile was observed. There was no obvious eGFP expression on days 1 and 2 after transduction, probably because of the slow process of Rep-mediated chromosome integration. Without any drug selection pressure, transgene expression began to appear on day 3; the population of eGFP-positive hES cells reached around 2% on day 7 as estimated under fluorescence microscope (data not shown). This percentage could be a rough estimation of the integration rate of our hybrid vectors in the original hES cell population. It also reflects the number of stable integrants because at this time point the native baculovirus vector-mediated transient gene expression has already been silenced. Thus, the hybrid vector-mediated integration rate is much higher than that produced by chemical-based, plasmid transfection method but still lower than that provided by lentivirus-based vectors. In the case of using eGFP as a reporter gene, this integration rate is already high enough to allow us to enrich those stable cells manually without the use of drug selection.
To increase the rate of stable integration of this hybrid system in hES cells, control of rep gene expression might be crucial. Active and consistent expression of the rep gene could result in chromosomal instability or mobilization of the transgene [17]. Several methods could be used to limit the duration and intensity of Rep expression, thus improving the efficiency of Rep-mediate chromosome integration. These include (a) expressing the rep gene from an inducible promoter to control the duration of Rep action, (b) delivering the rep gene into hES cells using a separate vector to manipulate the copy number of the rep gene transferred into one hES cell, and (c) direct delivery of the Rep protein rather than the rep gene into hES cells, in which the amount of the Rep protein in one hES cell can be controlled in a more precise way and the Rep-mediated integration would be terminated upon protein degradation. We are currently investigating these possibilities.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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We thank Dr. Jaana Jurvansuu for her critical review of the manuscript, Dr. Wang Chaoyang for his contribution to the construction of a hybrid baculoviral vector, and other lab members for helpful discussion and support. This work was funded by the Agency for Science, Technology and Research (A*STAR), Singapore to Institute of Bioengineering and Nanotechnology.
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Abstract
Introduction
