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School of Biological Sciences, Nanyang Technological University, Singapore
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Key Words. Site-specific recombination ? Human embryonic stem cells ? Cre recombinase ? integrase ? Plasmid transfection ? resolvase5 G( ^/ Y8 |4 k* q7 R
) P: O; h- ^) I' B% N7 U" U( XCorrespondence: Peter Dr?ge, 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@ntu.edu.sg, X, {: w$ Z9 s3 J2 c
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ABSTRACT
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" e) S3 z9 n9 i1 qHuman 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 . The great potential of hESCs in basic research, regenerative medicine, and gene therapy is apparent . 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.
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It has been noted that plasmid transfection is rather inefficient with hESCs . 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 ." |* x5 g u4 @+ \5 b0 o6 i
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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 . Furthermore, the Cre, Flp, C31, and integrase system can achieve targeted insertion of foreign DNA into predetermined artificial or natural genome sequences . 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 . A main objective, therefore, is to develop SSR systems that can be used to safely modify hESC genomes for possible clinical applications.' Q$ v+ b3 O0 Q3 X- b5 _& z
( o1 s/ }. D) I$ HAs a first step toward this goal, we compared the enzymatic activities of three site-specific recombinases: wild-type Cre, a modified resolvase (102NLS) derived from bacterial transposon bearing the two recombination-activating mutations E102Y and E124Q, and Int-h/218, which is a mutant phage integrase (E174K/E218K) that functions in the absence of cofactors in mammalian cells . 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 . 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.
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* n, `3 U- c2 [6 K0 \! K: K& X" @- UMATERIALS AND METHODS
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hESC Pluripotency on Matrigel
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7 X4 o5 {/ n- r* S7 F! uIt was demonstrated previously that some hESC lines can be grown on Matrigel in the presence of CM obtained from MEF cell cultures . 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–1F). This indicates that they remain pluripotent under these culture conditions, which are used for plasmid transfection and recombination assays below.
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~$ X9 p3 O0 x' ^# Y0 ~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.8 m# a0 p/ @+ |; F) b
k& o: I) E1 q! e3 g9 S2 ehESC Transfection Efficiency- O$ |+ A' w) L- h8 o' f0 u
+ P6 J" c" J" ?4 }" aUnlike mouse ESCs, hESCs are rather difficult to transfect. Protocols using lentiviral vectors , electroporation , and various transfection reagents 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 . 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. 2A). 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. 2A). A similar finding has been reported with COS-7 and CHO cells . 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.$ X; f) D: L! b( l8 x) o6 {9 @
4 U& o" I" h+ |9 s3 F1 s$ Q+ ^4 m9 v: o# WIn 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, 2C). 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 .+ D1 _. o4 b8 [+ ~! Z) q( T! ^0 N
" v1 N7 t4 t& x( O8 {SSR in hESCs0 g* t4 M7 E8 d; R$ ]/ A4 f
2 {, N; S$ i8 D' ?We recently developed two new SSR systems for applications in eukaryotic cells and showed that mutant phage integrases and the mutant resolvase 102NLS, in particular, are recombination-proficient on episomal and genomic DNA substrates . However, because hESCs differ from other mammalian cells, including mouse ESCs, in features such as cell size, doubling time, and gene expression patterns , we were interested in comparing the and 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. 3).! ^2 T9 i- T- a) ?. P7 d% K
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Figure 3. Recombination substrate vectors. (A): pCH-RLNRLE was used as substrate for Cre and resolvase. Recombination between the directly repeated 114-bp Res sites as target sequence for 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): pIR contains directly repeated attachment sites attB and attP as target sequences for the integrative pathway. pER 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.% }0 r" S/ p. e+ P
4 L9 K, |8 `7 m! g3 c9 @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 resolvase (taken as 100% recombination efficiency). We found that Cre recombined pCH-RLNRLE in more than half of the transfected cells (Fig. 4). In contrast, only 5% of transfected cells displayed detectable EGFP expression due to recombination of pCH-RLNRLE by resolvase. The latter finding is in agreement with previous results which showed that resolvase is almost as efficient as Cre in murine cells, but significantly less so in human cells . 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.4 t2 J7 h' a- v9 N2 F) a7 m y& w5 o
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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 resolvase and Cre were repeated four times. Those with 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
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In a second series of assays, we used pCMVssEGFP together with pCMV mock vector as positive control and demonstrate that Int-h/218 recombined the substrate for integrative recombination, pIR, in 20% of transfected cells (Fig. 4), which is similar to what we described for HeLa cells . Surprisingly, this number dropped to approximately 4% with pER, the substrate for excisive recombination. This deviates from our previous findings with HeLa cells in which this difference is less than twofold . 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 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.7 ?# [5 Z$ i5 Z
) B- \$ Y# d% C- YOur 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 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 102NLS in human cells. We can exclude differences in nuclear localization because both Cre and 102NLS contain functional nuclear localization signals . One possible factor to consider may be protein modification of resolvase in human cells.
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% Q( x4 E7 w* v4 _' o- gThe integrative pathway catalyzed by Int-h/218 is quite efficient in hESCs. Furthermore, because of the directionality that 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 integrase–mediated recombination genome insertions have been reported with human Burkitt’s lymphoma cells and mammalian artificial chromosomes .
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& Q/ @, k3 ?; E$ m# k, R7 m" aCurrently, 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 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 system in mammalian cells (Dr?ge, 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.8 m6 Y. C# j$ r/ a- M# K$ |3 R9 V! X
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发表于 2009-3-5 10:49
发表于 2015-5-21 19:15
