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First published online June 13, 2005
Stem Cells Vol. 23 No. 7 August 2005, pp. 868 -873
doi:10.1634/stemcells.2005-0044; www.StemCells.com
© 2005 AlphaMed Press

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Comparative Analysis of Sequence-Specific DNA Recombination Systems in Human Embryonic Stem Cells

Shen Mynn Tan, Peter Dröge

School of Biological Sciences, Nanyang Technological University, Singapore

Key Words. Site-specific recombination • Human embryonic stem cells • Cre recombinase • {lambda} integrase • Plasmid transfection • {gamma}{delta} resolvase

Correspondence: Peter Dröge, Ph.D., Nanyang Technological University, School of Biological Sciences, 60 Nanyang Drive, 637551, Singapore. Telephone: 65-6316-2809; Fax: 65-6791-3856; e-mail: pdroge{at}ntu.edu.sg


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The great potential of human embryonic stem cells (hESCs) in basic research, regenerative medicine, and gene therapy is widely recognized. Controlled manipulation of hESC genomes through sequence-specific DNA recombination (SSR) may play a significant role in future hESC applications. However, very little is known about the functionality of SSR systems in hESCs. We demonstrate here that mutant phage {lambda} integrase, phage P1 Cre recombinase, and mutant {gamma}{delta} resolvase displayed distinct activities on episomal recombination substrates. Interestingly, cofactor-independent {lambda} integrase catalyzed the integrative pathway five times more efficiently than the excisive pathway. Such a degree of directionality in hESCs could be explored for sequential gene insertions into predetermined genomic sequences. We also report an improved, easy-to-use plasmid transfection system that employs silica microspheres and, in combination with SSR, could be applied to hESC genome engineering.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Human embryonic stem cell (hESC) lines are derived from the inner cell mass of the early preimplantation embryo. These cells are genetically unaltered and remain pluripotent in culture over many cell generations [1–3]. The great potential of hESCs in basic research, regenerative medicine, and gene therapy is apparent [4]. To fully explore their potential, however, certain experimental techniques need to be improved or newly developed. This includes gene transfer technologies and tools for stable genetic modifications of hESCs.

It has been noted that plasmid transfection is rather inefficient with hESCs [5]. In addition, published data comparing different transfection strategies are scarce. This is especially true for reagent-based methods. We address this issue and directly compare transfection efficiencies obtained with different reagents. We also describe an efficient three-component plasmid transfection system for hESCs which employs silica microspheres [6].

Site-specific DNA recombination (SSR) systems derived from prokaryotic cells are valuable tools for various applications in eukaryotic cells. Notably, the phage P1 recombinase Cre is frequently used to splice out (delete) marker genes from genomes after gene targeting and for conditional mutagenesis in model organisms, particularly in the mouse [7]. Furthermore, the Cre, Flp, {Phi}C31, and {lambda} integrase system can achieve targeted insertion of foreign DNA into predetermined artificial or natural genome sequences [8–10]. The latter application is especially relevant to future gene therapy approaches with hESCs because it minimizes the risk of unwanted genome alterations due to random DNA insertions, which have been reported for viral vector-based strategies [11]. A main objective, therefore, is to develop SSR systems that can be used to safely modify hESC genomes for possible clinical applications.

As a first step toward this goal, we compared the enzymatic activities of three site-specific recombinases: wild-type Cre, a modified {gamma}{delta} resolvase ({gamma}{delta}102NLS) derived from bacterial transposon {gamma}{delta} bearing the two recombination-activating mutations E102Y and E124Q, and Int-h/218, which is a mutant phage {lambda} integrase (E174K/E218K) that functions in the absence of cofactors in mammalian cells [12–17]. It has been shown that these enzymes faithfully catalyze DNA strand transfer on their respective target sequences inside mammalian cells without adding or deleting nucleotides to or from strands in the course of the reaction, respectively [12, 13, 15, 17]. We show here that these recombinases catalyzed DNA strand transfer reactions inside hESCs on plasmid substrates after cotransfection with the respective expression vector. Whereas Cre-mediated recombination is detectable in approximately 50% of transfected cells, the integrative recombination pathway catalyzed by Int-h/218 is observed in close to 20% of transfected cells. Interestingly, the excisive recombination pathway is significantly less active in hESCs than the integrative pathway. This finding will be explored to achieve controlled site-specific gene insertions into predetermined chromosomal sequences.


   MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell Culture
hESC line hES2 (46XX; ES Cell International [ESI], Helios, Singapore, http://www.escellinternational.com) was cultured on ESI murine embryonic fibroblast (MEF) feeder cells according to previously established protocols [2, 18–19]. Prior to transfection, individual colonies were manually dissociated with a 27-gauge needle (Sigma, St. Louis, http://www.sigmaaldrich.com) into pieces of approximately 5 x 103 cells each. Four pieces were transferred into each well of a four-well plate (Nunc, Rochester, NY, http://www.nuncbrand.com) coated with Matrigel (BD Biosciences, Singapore, http://www.bdbiosciences.com) and cultured in MEF conditioned media (CM) as described [20–22]. Briefly, stock Matrigel solution was diluted 1:20 in ice-cold Dulbecco’s modified Eagle’s medium (DMEM) and incubated on plates at 37°C overnight. CM was prepared by collecting day-old hESC media from inactivated MEFs and filtered through a 0.2-µM syringe filter (BD Biosciences).

Transfection and Recombination Assays
hESCs were transfected 4 days after colony transfer. Approximately 2 x 105 cells in each well were transfected using Exgen 500 (Fermentas, Hanover, PA, http://www.fermentas.com), Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com), Fugene 6 (Roche, Basel, Switzerland, http://www.roche.com), Effectene (Qiagen GmbH, Hilden, Germany, http://www.qiagen.com), or Effectene plus 0.15 µM silica microspheres (Polysciences, Inc., Warrington, PA, http://www.polysciences.com). Transfections using Exgen 500 and Lipofectamine 2000 were performed as described [5, 23]. For Fugene 6, hESC media without serum was mixed with either 30 µl (for 2 µg DNA) or 60 µl (for 4 µg DNA) of reagent to yield a total volume of 100 µl and incubated at room temperature (RT) for 5 minutes. The appropriate amount of DNA was added to the mixture, incubated for 45 minutes, and subsequently added drop-wise to each well. Transfection using Effectene was performed as recommended by the manufacturer at a 1:25 DNA:Effectene (µg:µl) ratio. The final mixture was incubated for 15 minutes at RT before application. Transfection with Effectene and silica microspheres was performed as above, but with the addition of 1 x 109 microspheres 5 minutes after the start of the final incubation period. The reagent/DNA mixtures were incubated with hESCs for 24 hours.

Transfection efficiencies were determined with 2 µg of reporter plasmid pCMVssEGFP per well. Recombination assays were carried out with 2 µg of substrate vectors pCH-RLNRLE, p{lambda}IR for {lambda} integrative, or p{lambda}ER for {lambda} excisive recombination. Substrate vectors were cotransfected with 2 µg of respective recombinase vectors pPGKCre, pPGK{gamma}{delta}102NLS, or pCMVssInth/218 per well. pPGK or pCMV served as mock expression vectors. pCMVssEGFP was used as positive control for {lambda} integrative and excisive recombination, whereas plasmid pCH-RLE, the product vector that results from recombination on pCH-RLNRLE, served as positive control for Cre and {gamma}{delta} resolvase reactions. {lambda} integrase recombination assays were performed using Fugene 6. Cre and {gamma}{delta} resolvase assays were carried out with Effectene.

Immunostaining
Immunostaining of hESC colonies on Matrigel was performed using SSEA-1, SSEA-4, TRA-1-61, TRA-1-80, and OCT4 specific primary antibodies (Chemicon, Temecula, CA, http://www.chemicon.com). Antibodies were visualized with fluorescein iso-thiocyanate (FITC)–labeled secondary antibodies (Chemicon). Alkaline phosphatase (AP) activity was detected according to the manufacturer’s protocol (Chemicon).

Flow Cytometry
Cells were washed 48 hours after transfection with phosphate-buffered saline (PBS) (Gibco, Invitrogen Corporation) and harvested through incubation with 0.05% trypsin/ethylenediaminetetraacetic acid (EDTA) for 5 minutes. Cells were collected, washed once with PBS, and resuspended in 200 µl of PBS for analysis in a FACSCalibur flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). A minimum of 20,000 total events were recorded per sample and analyzed using CELLQuest software (Becton, Dickinson and Company). All values were obtained after gating mock transfected (pCMV) hESCs to zero. Recombination efficiencies were calculated after subtracting mock control values from experimental values. The positive control values were taken as 100%.

Substrate and Expression Vectors
Plasmids pCMV, pCMVssInt-h/218, pCMVssEGFP, pCH-RLN-RLZ, p{lambda}IR, p{lambda}ER, pPGKCre, and pPGK{gamma}{delta}102NLS have been described [13–16]. pCH-RLNRLE was constructed by inserting the coding region for enhanced green fluorescent protein (EGFP) into pCH-RLNRLZ. The EGFP gene was derived from pCMVssEGFP via polymerase chain reaction (PCR), with a short linker (GGSGG) replacing the EGFP start codon. The following primers were used in the PCR: L1EGFP-F (5'-CGGGGTACC-GGGTGGAAGCGGCGGTGTGAGCAAGGGCGAGGA-3') and L1EGFP-R (5'-CGCGGATCCGAGGCTAGAACTAGTGG-3'). Both the PCR product and vector pCH-RLNRLZ were cleaved with KpnI/BamHI. The EGFP insertion replaced a large portion of the LacZ gene and results in the expression of an N-terminal LacZ-Linker-EGFP fusion protein. The recombined product vector pCH-RLE served as positive control and was generated through transformation into 294-Cre cells. This Escherichia coli strain expresses Cre constitutively at a low level [24].


   RESULTS AND DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
hESC Pluripotency on Matrigel
It was demonstrated previously that some hESC lines can be grown on Matrigel in the presence of CM obtained from MEF cell cultures [20–22, 25]. We show that ESI cell line hES2 can also be cultured under these conditions and displays surface markers SSEA-4,TRA-1-60, and TRA-1-81. In addition, hES2 cells stain positive for AP and OCT4, but negative for SSEA-1 (Figs. 1A–1FGo). This indicates that they remain pluripotent under these culture conditions, which are used for plasmid transfection and recombination assays below.



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  		Figure 1. Immunostaining of hESC colonies on Matrigel. To assess pluripotency after transfer to Matrigel, hESC colonies were analyzed for expression of TRA-1-60 (A), OCT4 (B), SSEA-1 (C), TRA-1-81 (D), SSEA-4 (E), and AP (F), as described in Materials and Methods. Bars = 100 µm. Abbreviations: AP, alkaline phosphatase; hESC, human embryonic stem cell.

 
hESC Transfection Efficiency
Unlike mouse ESCs, hESCs are rather difficult to transfect. Protocols using lentiviral vectors [26, 27], electroporation [28, 29], and various transfection reagents [5, 23, 30] have been developed, but comparative analyses are scarce. Currently, lentiviral-based approaches appear to be the preferred choice to achieve high transfection efficiencies. However, these vectors are quite difficult to generate and have size limitations for transgenes [23]. Here we compare plasmid transfection efficiencies obtained with more popular reagents and demonstrate that hES2 cells exhibited a clear preference for lipid-based transfection reagents (Fig. 2AGo). Optimized Fugene 6, Effectene, and Lipofectamine 2000 showed similar transfection efficiencies of 5%. This number decreased to less than 1% with the polymer-based reagent Exgen 500. We also tested the addition of silica microspheres to lipid-based transfection reagents and found that it resulted in a close to threefold increase in plasmid uptake in combination with Effectene (Fig. 2AGo). A similar finding has been reported with COS-7 and CHO cells [6, 31]. On the other hand, addition of microspheres showed only a limited effect with Lipofectamine 2000 and none with Fugene 6 (data not shown). Improved DNA uptake through silica microspheres is based on the theory that DNA/ reagent/silica complexes increase the DNA concentration at the cell surface. This dependency on local DNA concentration might also lead to rather large variations between experiments.



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  		Figure 2. (A): Comparative analysis of different transfection reagents. hESCs were transfected with pCMVssEGFP, using various transfection reagents as indicated. The efficiency was determined via FACS as the percentage of cells expressing EGFP after gating pCMV-transfected cells as negative control. Each experiment was repeated three times, and the mean values with SDs are shown. (B, C): EGFP-transfected hESC colonies. (B): A large EGFP-transfected colony, with many EGFP-expressing cells in the monolayered periphery, but none in the thick, clustered center. Bar = 250 µm. (C): The smaller colony displayed has EGFP-expressing cells evenly distributed. Twofold higher transfection efficiencies (more than 20% from preliminary results) can be obtained if the transfected colonies are monolayered and small. Bar = 100 µm. Abbreviations: EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting; hESC, human embryonic stem cell.

 
In addition to being a rather efficient transfection reagent in combination with silica microspheres, Effectene also showed the least cytotoxicity based on FACS (fluorescence-activated cell sorting) flow rates (data not shown). Moreover, transfection of smaller, monolayered colonies could further enhance DNA uptake at least twofold (Figs. 2B, 2CGo). This is most likely due to more cells being directly exposed to DNA-bearing complexes. Our plasmid transfection efficiencies in the range of 10%–20% using the commonly available and easy-to-use Effectene in combination with silica microspheres are comparable to those achieved with more sophisticated protocols, such as nucleofection [29].

SSR in hESCs
We recently developed two new SSR systems for applications in eukaryotic cells and showed that mutant phage {lambda} integrases and the mutant {gamma}{delta} resolvase {gamma}{delta}102NLS, in particular, are recombination-proficient on episomal and genomic DNA substrates [13–17]. However, because hESCs differ from other mammalian cells, including mouse ESCs, in features such as cell size, doubling time, and gene expression patterns [32, 33], we were interested in comparing the {lambda} and {gamma}{delta} SSR system with the widely used Cre system inside hESCs. For this, we used substrate vectors that, when cotransfected with the respective recombinase expression vector, lead to EGFP expression as an easy read-out for recombination (Fig. 3Go).



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  		Figure 3. Recombination substrate vectors. (A): pCH-RLNRLE was used as substrate for Cre and {gamma}{delta} resolvase. Recombination between the directly repeated 114-bp Res sites as target sequence for {gamma}{delta} resolvase, or between the directly repeated 34-bp Lox sites as target sites for Cre, leads to the excision of the neomycin resistance gene (Neo). This deletion results in EGFP expression from the N-terminus-LacZ-EGFP–fused coding region. (B): p{lambda}IR contains directly repeated attachment sites attB and attP as target sequences for the {lambda} integrative pathway. p{lambda}ER instead contains attL and attR as direct repeats that serve as target sequences for the excisive pathway. Recombination between attB and attP or between attL and attR deletes the promoter-neomycin-transcriptional stop (PGK-Neo-TSS) cassette and results in EGFP expression then driven by the CMV promoter. Abbreviations: CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein.

 
In the first series of recombination assays, we used recombined product vector pCH-RLE and pPGK as positive control for recombination catalyzed by Cre and {gamma}{delta} resolvase (taken as 100% recombination efficiency). We found that Cre recombined pCH-RLNRLE in more than half of the transfected cells (Fig. 4Go). In contrast, only 5% of transfected cells displayed detectable EGFP expression due to recombination of pCH-RLNRLE by {gamma}{delta} resolvase. The latter finding is in agreement with previous results which showed that {gamma}{delta} resolvase is almost as efficient as Cre in murine cells, but significantly less so in human cells [34]. We want to emphasize that we are unable to determine recombination efficiencies at plasmid level because the copy number of internalized substrate DNA molecules accessible for the recombinase is unknown. This number varies per cell, and it is reasonable to assume that a single recombined copy will suffice to produce an EGFP-positive cell. Hence, the actual percentage of recombined substrate molecules could be lower than the percentage of EGFP-expressing cells normalized to the positive control.



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  		Figure 4. Comparative analysis of three site-specific recombination systems in human embryonic stem cells. Cells were cotransfected with substrate vector and the respective recombinase expression vector. In each case, the recombination efficiency was determined 48 hours after transfection. Experiments with {gamma}{delta} resolvase and Cre were repeated four times. Those with {lambda} integrase were repeated three times. The mean values with the SDs are shown. Based on the two-tailed paired Student’s t-test, all values are significant compared with respective negative controls, and p values are always < .05.

 
In a second series of assays, we used pCMVssEGFP together with pCMV mock vector as positive control and demonstrate that {lambda} Int-h/218 recombined the substrate for integrative recombination, p{lambda}IR, in 20% of transfected cells (Fig. 4Go), which is similar to what we described for HeLa cells [15]. Surprisingly, this number dropped to approximately 4% with p{lambda}ER, the substrate for excisive recombination. This deviates from our previous findings with HeLa cells in which this difference is less than twofold [15]. Because integrative recombination between attachment sites attB and attP generates sites attL and attR, which are then substrates for excisive recombination, our results imply that cofactor-independent {lambda} integrases display a significant degree of directionality inside hESCs. The molecular basis for this pronounced directionality in hESCs is not understood at the moment and deserves further investigation.

Our finding that the Cre recombinase is able to recombine episomal substrates in a significant fraction of cotransfected cells indicates that this recombinase is a good candidate for future hESC genome manipulations such as removal of marker genes. However, in our direct comparison with Cre, the resolvase mutant {gamma}{delta}102NLS is significantly less active in hESCs than in CHO cells and, therefore, may be useful only for specific applications that do not require high recombination efficiencies. It is not clear at present what causes the reduced activity of {gamma}{delta}102NLS in human cells. We can exclude differences in nuclear localization because both Cre and {gamma}{delta}102NLS contain functional nuclear localization signals [16, 35]. One possible factor to consider may be protein modification of {gamma}{delta} resolvase in human cells.

The {lambda} integrative pathway catalyzed by Int-h/218 is quite efficient in hESCs. Furthermore, because of the directionality that {lambda} Int-h/218 exhibited in hESCs, in particular, this SSR system could prove very valuable for targeted gene insertions into genomic attachment sites. These sites could be artificially introduced or occur naturally in the hESC genome. Examples for {lambda} integrase–mediated recombination genome insertions have been reported with human Burkitt’s lymphoma cells and mammalian artificial chromosomes [9, 10].

Currently, we are evaluating experimentally more than 1,000 different human genome sequence tracts as potential target sites that are homologues to the 21-bp attB site. These tracts contain inverted consensus {lambda} integrase recognition sites separated by a 7-bp–long spacer. Nearly all of these genomic sequences are single copy because of a unique spacer. Based on the high fidelity of the {lambda} system in mammalian cells (Dröge, unpublished results), it should be possible to specifically direct the insertion of vectors into a few selected sites that may be more accessible in hESC chromatin. Because a strategy of gene insertion employing the pair attB/attP, or derivatives thereof, will generate attL/attR sites in the genome, integration could be stable even in the continued presence of the recombinase. This should allow for sequential targeting events into different genomic loci.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 
We thank Curt Davey and members of our laboratory for critical comments on the manuscript. We acknowledge the financial support of A*STAR, Singapore, and Nanyang Technological University.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Received on January 31, 2005; accepted for publication on May 11, 2005.




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