Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome
© Xu et al.; licensee BioMed Central Ltd. 2013
Received: 5 June 2013
Accepted: 9 October 2013
Published: 20 October 2013
Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays.
We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase.
We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase.
KeywordsSerine recombinases Genome manipulation DNA damage
Serine integrases are phage-encoded site-specific recombinases that promote conservative recombination reactions between short (40-60 bp) DNA substrates located on the phage (phage attachment site, attP) and bacterial (bacterial attachment site, attB) chromosomes . The product of attP × attB recombination is an integrated prophage flanked by two new recombination sites, attL and attR, each containing half sites derived from attP and attB. In the absence of accessory factors the integrases mediate unidirectional recombination between attP and attB with greater than 80% efficiency. In the presence of a phage-encoded accessory protein, the recombination directionality factor (RDF) the attP × attB recombination is inhibited and the attL × attR recombination is stimulated [2, 3]. In this way integration (attP × attB) and excision (attL × attR) of the phage genome are under strict controls and in tune with the phage life cycles. The unidirectional activity, short substrate sites and functional autonomy of these recombinases has made them a useful complement to the widely used reversible recombinases of the tyrosine recombinase family such as Cre and Flp for genome engineering reviewed in . In particular the unidirectional activity has made them valuable for the promotion of DNA integration by recombinase-mediated cassette exchange reactions and for the development of iterative recombination approaches [4–7]. To date five serine integrases derived from phages φC31 , φBT1 , Bxb1 [10, 11] and R4 [11, 12] have been shown to be capable of promoting site-specific integration of DNA into mammalian genomes while TP901-1 , A118, FC1 and φRV  have been shown to promote site-specific recombination in an extra-chromosomal environment in mammalian cells. With one exception these studies have, however, been carried out largely independently of one another, in different cell lines, cells of different species and using different protocols. The exceptional study was that of Yamaguchi and colleagues  who compared the activities of the φC31, Bxb1, TP901-1 and R4 integrases in mediating site-specific recombination into a human artificial chromosome (HAC) isolated in hamster cells. This study exploited a promoter trap strategy and thus relied upon selection to assay recombination products. Importantly, however, the products were not analyzed at the level of DNA sequence. It was therefore neither possible to determine the total level of recombination promoted by these different enzymes nor to determine the fraction of recombination events that had proceeded by reciprocal and conservative site-specific recombination.
The discovery that site-specific recombination mediated by the φC31 integrase is sometimes accompanied by DNA damage in vertebrate cells identified  posed the question as how far integrase associated DNA damage limits the use of the serine integrases as genome engineering reagents. Damage of the type seen with the φC31 integrase, has not been detected with the tyrosine recombinases, is likely to be a consequence of the DNA cleavage and strand exchange mechanism of the serine recombinases (reviewed in ). The recombination pathway begins with binding of integrase to the attachment sites, which are then brought together by protein:protein interactions to form a synaptic tetramer. The reaction then proceeds by the formation of concerted double strand breaks in both of the DNA substrates prior to subunit rotation and recombination. It seems likely that the damage that accompanies the activity of these recombinases in vertebrate cells arises as a consequence of these double strand breaks being recognized by the mammalian cell double strand break repair pathways. It should be noted that to date no damage has ever been observed to accompany the action of the serine integrases in bacteria and it may therefore be the eukaryotic chromatin environment or the nature of the mammalian repair pathways that leads to the damage seen in mammalian cells. The frequency and extent of the damage would seem likely to reflect the time spent by the integrases in the covalently linked, cleaved DNA complex as this is most likely to be the target for the repair pathways.
In total there are fifteen phage-encoded serine integrases for which both of their attachment sites are known. Nine of these fifteen integrases have been characterised in reactions in mammalian cells, E. coli or in vitro (φC31 , Bxb1 , φBT1 , φC1 [14, 20], MR11 , TP901-1 , R4 , A118 , and φRV , ) while six (TG1, φ370.1 , Wβ , BL3, SPBc and K38) have not yet been shown to be active outside their native hosts. In total there are ten integrases whose utility as tools for integrating DNA into mammalian genomes has not been investigated. We have therefore set out to rank the activities of all fifteen of these serine integrases for which the sites are known by the criteria of both accuracy and efficiency in two different mammalian cell lines; human HT1080 cells and mouse ES cells. These studies have provided us with a clear rank order for the utility of this important class of enzymes as tools for vertebrate genome engineering with Bxb1 integrase mediating the most efficient and accurate site-specific recombination in this heterologous environment. The φC31 integrase comes a close second. For the remaining integrases, we demonstrate that DNA damage is an important factor in limiting the utility of members of this class of enzymes for promoting integration reactions in mammalian cells.
Results and discussion
Fifteen unidirectional 'phage integrases are active in E. coli
In vivo recombination activity after overexpression of integrases in E. coli BL21(DE3)
μg tagged protein/ml1
Assaying the unidirectional phage integrases in vertebrate cells
In both assays the integrases were expressed using plasmids (Figure 2E) in which each integrase gene had been codon optimized and tags placed at the 5′ and 3′ ends encoding the StrepII tag and the nuclear localization signal. An internal ribosome entry site (IRES) followed by an antibiotic resistance gene was placed downstream of each integrase gene.
Identification of eight unidirectional phage integrases promoting site-specific deletion in mammalian cells
Firstly we wanted to determine which of the fifteen integrases were active on substrates integrated into vertebrate genomes. The most sensitive way in which to detect activity is to assay recombination between attB and an attP sites in cis because the proximity of the sites favours the kinetics of synapsis, the process by which integrase bring the substrates together in a tetramer prior to DNA cleavage. We therefore transfected the construct designed to assay deletion, attP array CCAG HyTK attB array, into human HT1080 cells by electroporation and selected for stable transfectants using hygromycin. We screened 96 stably transfected clones for the integrity of the integrated DNA using PCR, checked for single copy integrants by restriction enzyme analysis and filter hybridization and then confirmed the integrity of the attP and attB arrays by sequencing. In this way we recovered four independent, stably transfected clones from two independent transfections, each containing a single copy of the integrated deletion reporter construct, attP array CCAG HyTK attB array.
In order to compare the activities of the different integrases we first transiently transfected 105 HT1080 cells containing the attP array CCAG HyTK attB array reporter with expression plasmids for each of the integrases using Lipofectamine and assayed for recombination activity by selecting for resistance to gancyclovir, a drug which is selectively toxic for cells expressing the HyTK fusion. None of the integrases gave a significant increase in the number of gancyclovir-resistant cells as compared to the empty expression vector (Adiitional file 1: Table S3) and so we assayed pools of the resistant cells for the presence of the recombinant attR site using PCR. Recombination activity was detected in populations of cells transfected with the R4, φC31, φBT1, Bxb1, SPBC and Wβ integrase expression constructs (not shown). However the low level of activity overall made it impossible to conclude anything about the integrases that did not yield attR in the PCR reactions as the integrases may be active but causing damage that removed the attR primer binding sites, may simply be slow in promoting site-specific recombination or completely inactive. Moreover the variability of the relative numbers of gancyclovir resistant clones generated in different experiments made an accurate comparison between the active integrases impractical.
Accuracy of integrase mediated site-specific recombination in human HT1080 and mouse ES cells
del clone 1 DattR
del clone 2 DattR
del clone 3 DattR
del clone 4 DattR
del clone 5 DattR
del clone 6 DattR
del clone 7 DattR
ES del clone 1 DattR
intn clone 1-3 DattR
intn clone 1DattR
ES del clone 1 DattR
ES del clone 2 DattR
del clone 1 DattR
We wanted to know whether the complete failure to detect deletion activity after two weeks of growth in the presence TP901-1, FC1, φ370.1, K38, φRV, A118, BL3 and MR11 integrases was because these recombinases were slow or because they were damaging the target sites. We therefore applied hygromycin selection to the second group of clones that had been stably transfected with the integrase expression construct but not exposed to gancyclovir, then relaxed hygromycin selection and analysed the clones for any detectable recombination after a further two weeks by PCR. The results were clear; TP901-1 integrase showed detectable recombination after further culture indicating that it was indeed slow but the remaining integrases showed no evidence of recombination (Additional file 1: Table S4). As before we determined the sequence of the PCR products containing the predicted attR site generated by the TP901-1 integrase; 4 were intact attR sites and 1 was damaged thus showing that TP901-1 integrase is like the R4 integrase and prone to site damage. We wanted to know whether the remaining 8 integrases that had failed to show productive recombination were attempting recombination but were in fact damaging the attachment sites. We therefore sequenced their substrate attachment sites (attP and attB) in the integrated reporter plasmids in two of the hygromycin resistant clones that had been transfected with the respective integrase expression constructs. None showed evidence of site damage.
Thus we concluded that the R4, φC31, φBT1, Bxb1, SPBc, TP901-1 and Wβ integrases are active on substrates integrated in to the genome of HT1080 cells although the TP901-1 integrase promotes recombination slowly and that the FC1, φK38, RV, A118, BL3 and MR11 integrases are not detectably active on integrated substrates. We cannot make any statement about the TG1 and φ370.1 integrases because we were unable to recover clones expressing these integrases for experimental analysis. We attempted to use western blotting to assay for expression of the integrases using with an antibody to the N-terminal StrepII tag but we were unable to detect any signal with any of the integrases suggesting that they are all expressed at low levels. Rank ordering of the deletion activities of the integrases in this experiment indicated the following Bxb1 = φC31 = φBT1 > R4 = Wβ > SPBc > TP901-1. The utility of the R4 integrase would seem to be limited by its liability to site damage. The purpose of this part of our project was to identify those enzymes that were active in vertebrate cells and we did not investigate the background of GANCrattR - clones. Although such clones were seen in the cells transfected with the empty vector and may arise from background silencing of the attP CCAG HyTK attB indicator gene or from loss of the chromosome carrying this gene, they occur at a higher level in the clones that had been transfected with integrases (Additional file 1: Table S4). We cannot exclude the possibility that they arise as a result of recombinase-mediated target site damage, although the accurate recombination activities seen with five of the seven active integrases would suggest that this is unlikely. The source of these background clones is therefore unclear.
It is also clear that not all of the clones that were successfully transfected with a construct expressing an active integrase yielded recombinant products. Thus even with the Bxb1 and φC31 integrases about 30% of the clones that were resistant to the antibiotic used to select for the presence of the expression construct failed to yield clones that were resistant to gancyclovir and contained an attR site. One hypothesis was that these clones failed to express sufficient integrase to bind and synapse the substrates, processes that are dependent on the affinity of integrase for its attP and attB sites and the expression level of integrase. We tried to test this idea by using the StrepII epitope with which we had tagged all of the integrases but, as before, were unable to do so for all of the integrases because the StrepII epitope tag was insufficiently sensitive and no signal was obtained in the western blots. However we had specific polyclonal antibodies for the φC31 and φBT1 integrases that we expected would be more sensitive and indeed they allowed us to measure the presence of the respective integrases by western blotting,. The results of this analysis was consistent with the notion that at least for some integrases the failure of site-specific recombination was associated with inadequate or low levels of expression (Additional file 1: Figure S2). The success of the western blotting using the polyclonal antibodies also demonstrates that the previous failure to detect integrase expression in the human cells using the StrepII tag and antibody was due to the relatively low sensitivity of this system.
The results obtained with the human HT1080 cells posed the question of whether they were generally true for vertebrate cells. Genome engineering of mouse embryonic stem cells (ES cells) is widely practiced and so we used a deletion strategy to assay the activities of the integrases in mouse ES cells. We used sequence targeting to introduce the attP array CCAG HyTK attB array deletion reporter cassette into the ROSA26 locus (Additional file 1: Figure S1) and then assayed for deletion by gancyclovir resistance following transient transfection with the set of integrase expression constructs described above. In order to avoid problems with toxicity that would compromise the practical significance of any results we chose to use transient assays and an internal control to carry out the experiment. In this ES cell system there appeared to be a clearer difference between the numbers of gancyclovir-resistant clones seen with the empty vector and those with expressing integrase than was observed in the HT1080 cells. The difference was not absolute however and identification of active integrases also required PCR analysis for the presence of a recombinant attR site. The results (Additional file 1: Table S5 and Figure 4B) were similar but not identical to those seen in the HT1080 cells; R4, φC31, φBT1, Bxb1, SPBc, Wβ and TG1 integrases showed detectable activity but the TP901-1 integrase did not. The activity seen with the TG1 integrase was, however, weaker than seen with the others with only one out of eight clones containing a detectable attR site. Rank ordering of the deletion activities of the integrases in this experiment indicated the following Wβ > Bxb1 > φC31 > SPBc > R4 > φBT1. We analyzed the accuracy of the site-specific recombination mediating the deletion reaction in two gancyclovir-resistant clones generated by the Bxb1, R4, Wβ and SPBc integrases. These results demonstrated that the Bxb 1, φBT1 and Wβ integrases all mediated site-specific recombination accurately but that the R4 integrases was associated with a deletion in one of the two recombination products analyzed and that both of the SPBc products were deleted. In the case of the SPBc integrase the region deleted 171 bp in the attP array extending as far as the φ370.1 attP site raising the possibility of a lack of specificity in attP site recognition by the integrase. This however seems unlikely because the breakpoint in the φ370.1 attP site is not at the proposed recombination junction but 6 bp 3′ of the point of symmetry defining the pseudo-palindrome of this attachment site.
The failure to detect recombinase activity mediated by the φ370, FC1, TG1, RV, FC1, φK38, MR11, A118 and BL3 integrases in HT1080 cells using deletion substrates integrated into the vertebrate genome posed the question as to whether these integrases were active in HT1080 cells or whether they were simply prevented from acting by the fact that the target sites were integrated into the genome. We therefore carried out a series of transient tranfection experiments in which the deletion substrate plasmid was transiently transfected into HT1080 cells that had been stably transfected with an expression plasmid for one of these integrases or, in the case of the φ370 and TG1 integrases,(where such stably transfected cell lines did not exist) co-transfected the respective expression plasmid and deletion substrate plasmid and then analyzed the extracted DNA for deleted plasmid after 72 hours by PCR. The recombinants were assayed using one of two PCR reactions for which the Bxb 1 or Wβ integrases acted as positive controls. The results (Figure 4C) demonstrate that the RV, φK38, MR11, A118 and BL3 integrases were in fact active in the HT1080 cells and thus we conclude that these integrases are unable to promote site-specific recombination when their substrates are integrated into the genome of the HT1080 cells but can do so when they are present extra-chromosomally. We can make no statement about the φ370, FC1, TG1 integrases as we have no evidence to determine whether they are expressed.
Comparative integration activities of seven unidirectional phage integrases in mammalian cells assayed by recombinase mediated cassette exchange
As before we also tried to assay the ability of these seven enzymes to mediate site-specific integration in mouse cells using transient expression of the integrase. None were detectably active suggesting that as in human cells detectable integration with this configuration of attachment sites requires stable expression of the integrase.
The major conclusion following from this work is that although all of the fifteen unidirectional serine integrases for which both attachment sites have been identified are active in E.coli, only four of these; Bxb1, φC31, R4 and φBT1 are able to mediate accurate site-specific integration into genomic DNA in human cells and rank such that Bxb1 is marginally better than φC31 and both are better than R4 and φBT1 integrases. The three integrases from the phages Wβ, SPBc, TP901-1 are active in vertebrate cells as judged by their ability to mediate site-specific deletion and by their ability to mediate integration reactions extra-chromosomally but fail to complete successful site-specific integration when attempting an integration by cassette exchange because they damage one or other of the participating DNA molecules. Although there are differences (Figure 4) between the activities seen with the different integrases in human and mouse cells the data the overall pattern of activites in the two cell types is such that it would seem prudent to adopt the B×b 1 integrase as the first choice in both. We have not investigated the causes of the different activities but they must reflect interactions between the respective integrases and host encoded proteins.
Our observations have three practical implications and pose one question. The practical implications are first that the B×b 1 integrase should be the first choice for any genome engineering in vertebrate cells that requires the use of a serine integrase. Second, that screening for more serine integrases that can be used in vertebrate cells is likely to have a low success rate and, third, that it is necessary that all site-specific integrants, particularly those generated by the R4 and φBT1 integrases should be checked for the fidelity of the recombination reaction.
If chromatin is inhibiting strand exchange by the serine integrases, one might also have reasonably expected chromatin to alter other activities of integrase such as site-selection. In vitro φC31 integrase only recombines attP × attB and is never active on other pairs of attachment sites including attP with attP or attB with attB. This site-selectivity is explained as only integrase dimers bound to attP and to attB can tetramerise to form the synaptic complex. Within this complex activation of DNA cleavage occurs. In all of our experiments we did not observed altered site-selectivity or DNA damage arising from some attempt at recombination of two attP sites or two attB sites. The absence of any change in site-selectivity in eukaryotic chromatin implies that the proposed conformational differences between integrase bound to an attP site and to an attB site are robust to withstand any fortuitous protein-protein interactions arising with chromatin.
The differences seen between the activities of the integrases in E. coli and in vertebrate cells on extra-chromosomal substrates on one hand and on substrates integrated into the genome on the other and the explanation for the site damage seen in the integration reactions both suggest that chromatin and other DNA binding proteins are important factors in limiting the activity of site-specific recombinases in vertebrate cells. It may therefore be of value in future experiments to determine how the activity of the integrases vary according to the position of the target sites in the genome.
Bacterial strains, plasmids and molecular biology
E. coli K12 DH5α was used as a general cloning host and propagated on LB medium containing appropriate supplements or antibiotics (ampicillin (100 μg/ml), chloramphenicol (50 μg/ml), X-gal (120 μg/ml), IPTG (40 μg/ml). E. coli BL21 (DE3) was used as a protein over-expression host. To construct the integrase expression plasmids, the integrase genes were amplified by PCR from either phage templates or from plasmids and inserted into pET21a vector (Novagen) using either the In-Fusion cloning system (Clontech) or T4 ligase and compatible restriction sites. The native sequences were used as the templates for the amplification of all integrase genes except for φC31, A118, FC1 and φK38 for which templates were derived from synthetic, codon optimised forms (Genescript). Each integrase gene was modified to include a StrepII tag at the 5′ end and a nuclear localisation sequence (NLS) at the 3′ end. Reporter plasmids containing cognate attP and attB sites for all the integrases were cloned into pACYC184. PCR was used to amplify the lacZα gene using forward and reverse primers that contained the attB and attP sites (both approximately 50 bp in length) in head-to-tail orientation (Additional file 1: Table S1). All of the constructed plasmids were verified by sequencing (Dundee Sequencing Service). The sequences of all PCR plasmids and primers used in the bacterial work are listed in Additional file 1: Table S1 and Additional file 1: Table S2.
Recombination assays in E. coli
The activities of the cloned integrases in E. coli were assessed by two assays, which relied on different expression regimes for the integrases. The expression vector pET21a has a T7 RNA polymerase promoter to drive the transcription of the integrase genes and this was used to ensure expression of the integrase genes in the E. coli host BL21(DE3). In the absence of T7 RNA polymerase, as is the case in E. coli DH5a, expression of integrase is dependent on the recognition of the T7 promoter by host RNA pol ymerase, which will result in lower expression.
DH5α cells containing the attB/attP reporter plasmids were transformed with the appropriate integrase expression plasmid and plated out on LB agar plates containing ampicillin (100 μg/ml), chloramphenicol (50 μg/ml), X-gal (120 μg/ml), IPTG (40 μg/ml) and incubated overnight at 37°C. Recombination between the att sites deletes the lacZα gene from the reporter plasmid leading to white colonies and therefore recombinants (white colonies) were scored amongst a background of non-recombinants (blue colonies). The transformation plates derived from several integrases gave rise to only blue or light blue colonies. Restreaking of these colonies was performed to determine if white colonies (containing recombinant reporter plasmids) could segregate.
BL21(DE3) cells containing the attB/attP reporter plasmids were transformed with the appropriate integrase expression plasmid and plated out on LB agar plates containing ampicillin and chloramphenicol. After an overnight incubation a single colony was picked and grown in 5 ml 2YT containing ampicillin and chloramphenicol for 5 h at 37°C. The cells were induced with IPTG and grown overnight at 20°C after which 1 ml of this culture was used to prepare plasmid and the pellet from 4 ml was used in Western blotting to estimate the expression of integrase. Plasmids extracted from the overnight culture were used to transform DH5α cells and colonies growing on plates containing chloramphenicol, X-gal and IPTG after overnight at 37°C were examined. The proportion of recombinant versus non-recombinant plasmids was scored from the proportion of white versus blue transformants. PCR was carried out on 5 individual colonies for each integrase used in the assay to amplify and sequence the attL sites, remaining in the recombinant plasmids. Cell pellets from the overnight cultures were resuspended in LB, incubated in SDS sample buffer and the proteins separated on a 4-12% SDS gel (Expedeon, UK). The integrase protein was detected by Western blotting using a monoclonal antibody against the StrepII tag conjugated to horseradish peroxidise (IBA, Germany). The amount of protein present was determined by comparing the intensities of the bands from the Western blotting to those from a dilution series of purified StrepII-tagged φC31 integrase. The intensity of the bands was determined using ImageJ (NIH) and the results were expressed as μg of total protein per ml culture.
In vitrorecombination assay
Integrase recombination activity was assayed in vitro using substrates in which the attachment sites were present either isolated from other attachment sites or within the arrays containing the attachment sites for all 15 integrases of interest. The combination of substrates was as follows: pRT702 (φC31 attP site) and pRT600 (φC31 attB site), pRT702 and pUC57_attB array, pUC57_attP array and pRT600, pUC57_attP array and pCCAG_attB array, pCCAG_attP array and pUC57_attB array. The substrates were incubated in recombination buffer (10 mM Tris pH 7.5, 100 mM NaCl, 5 mM DTT, 5 mM spermidine, 4.5% glycerol, 0.5 mg/ml BSA) and φC31 integrase (0 – 700 nM) for 1 h at 30°C  After heat inactivation at 80°C for 10 min the reaction was digested with HindIII or BamHI and the recombination molecules detected by gel electrophoresis. Gel images were analysed using ImageJ (NIH); the intensity of the bands was determined (after subtraction of base line intensities) and used to quantify the depletion of substrates and appearance of products.
Human cell culture
Human HT1080 fibrosarcoma cells were grown as described previously except that RPMI 1640 rather than Dulbecco’s modified Eagles Medium was used as the un-supplemented medium. G418, Hygromycin and gancyclovir (Invitrogen) were used at 400 mg/ml, 100 mg/ml and 20 μM respectively for selection. Cells were transfected with DNA either by electroporation using a BTX ECM 630 at 400 V, 50 Ω and 250 μF or using Lipofectamine (Invitrogen) as recommended by the manufacturers. We used electroporation with 500 μg of linearized DNA and zeocin selection for the stable introduction of the integrase expression constructs and lipofection of 1.6 μg of closed circular DNA for the transient introduction of either substrate or integrase expression construct.
ES cell manipulation and analysis
Targeting vector construction and gene targeting
The insertion of the attP-Hygro-TK-attP array at the ROSA26 locus was performed by gene targeting in E14tg2a ES cells (Additional file 1: Figure S1). A targeting vector for the ROSA26 locus, pRosa26.10  was adapted by the insertion of an AscI-NsiI-SacII polylinker, made by oligonucleotide annealing, into the AscI and SacII sites 3′ and 5′ of the homology arms, creating plasmid pRosa26-PL. The final targeting vector, pRosa26-PHB containing the attP-Hygro-TK-attB arrays, was constructed by inserting the arrays from pBSattPCCAGHyTKattB into pROSA26-PL via the 5′ AscI and the 3′ NsiI sites.
The targeting vector was linearized by XhoI digestion and electroporated into 1×107 E14tg2a cells at 500 V, 3 uF. Cells were plated on gelatin and recombinant clones were recovered by selection in Hygromycin 75 ug/ml. Targeted clones were identified by long range PCR screening using primers 5′-GGCACTACTGTGTTGGCGGA-3′ and 5′-GGCCAGCTTATCGATACCGT-3′ for the 5′ end; 5′-AGCGAGGGCTCAGTTGGGCTGTTT-3′ and 5′-CTCAGTGGCTCAACAACACTTGGTCA-3′ for the 3′ end. Single copy integration events were confirmed by Southern blotting using an EcoRV digest and an internal Hygromycin probe.
Transient assays of integrase-mediated deletion in ES cells
1 × 106attP-Hygro-TK-attB ES cells were electroporated with 5 μg of the integrase vectors and 200 ng of a control plasmid containing a functional neomycin cassette to control for transfection efficiency. Electroporation was performed using the Neon transfection system (Invitrogen) (3 × 1400 V, 10 ms). Cells were plated on 2 wells of a 6 well plate and selected in either 3 μM Gancyclovir or 350 μg/ul G418. On the 8th day of selection resistant colonies were stained in methylene blue and counted. Deletion efficiencies were calculated from the number of gancycolvir resistant colonies normalised against transfection efficiency by assessing the number of G418 resistant clones in the replica plating. All comparison transfection experiments were repeated twice using 2 independently targeted ES cell clones.
Individual gancyclovir resistant colonies were picked and expanded for the preparation of genomic DNA. PCR analysis using primers del-Rosa-F1 (5′ GATCGAGGTGCCCCAACTGGGGTAACCT TT-3′) and del-Rosa-R1 (5′-CCGCGGATGCATAGCGGATAACAATTTCAC-3′) which bind at the very 5′ and 3′ of the integrated attP-Hygro-TK-attB array and amplify across the expected deletion events (Additional file 1: Figure S1), was performed to confirm the deletion event had occurred. PCR products were sequences to assess the nature and position of the attP x attB recombination and the sequences of the resulting attR sites. Gancyclovir resistant clones that failed to yield an amplicon using this PCR strategy were further investigated for aberrant recombination events using primers which amplify the hygromycin sequence (5′- GAAGAATCTCGTGCTTTCAGCTTCGATG-3′; 5′- AATGACCGCTGTTATGCGGCCATTG-3′) and the thymidine kinase sequence (5′-TCTGGACCGATGGCTG TGTA-3′; 5′-AGACGTGCATGGAACGGAGG-3′).
This work was supported by BBSRC grant: BB/H005277/1; New recombinases for genome engineering.
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