Comparative analysis of right element mutant lox sites on recombination efficiency in embryonic stem cells
© Araki et al; licensee BioMed Central Ltd. 2010
Received: 10 October 2009
Accepted: 31 March 2010
Published: 31 March 2010
Cre-mediated site-specific integrative recombination in mouse embryonic stem (ES) cells is a useful tool for genome engineering, allowing precise and repeated site-specific integration. To promote the integrative reaction, a left element/right element (LE/RE) mutant strategy using a pair of lox sites with mutations in the LE or RE of the lox sequence has previously been developed. Recombination between LE and RE mutant lox produces a wild-type loxP site as well as an LE+RE double mutant lox site, which has mutations in both sides and less affinity to Cre, resulting in stable integration. We previously demonstrated successful integrative recombination using lox71 (an LE mutant) and lox66 (an RE mutant) in ES cells. Recently, other LE/RE mutant lox sites showing higher recombination efficiency in Escherichia coli have been reported. However, their recombination efficiency in mammalian cells remains to be analyzed.
Using ES cells, we compared six RE mutant lox sites, focusing on their recombination efficiency with lox71. All of the RE mutant lox sites showed similar recombination efficiency. We then analyzed the stability of the recombined product, i.e., the LE+RE double mutant lox site, under continuous and strong Cre activity in ES cells. Two RE mutants, loxJTZ17 and loxKR3, produced more stable LE+RE double mutant lox than did the lox66/71 double mutant.
The two mutant RE lox sites, loxJTZ17 and loxKR3, are more suitable than lox66 for Cre-mediated integration or inversion in ES cells.
Integrative recombination is useful for the production of transgenic animals or cells because any DNA of interest can be introduced into a chromosomally located lox site. However, integrative recombination between wild-type loxP sites is inefficient due to re-excision through intramolecular recombination . Studies of mutated loxP sites have revealed that two classes of mutations can promote Cre-mediated insertion or replacement. One class consists of heterospecific lox sites carrying mutation(s) in the central 8-bp spacer region [6–8]. Recombination does not occur between two lox sites differing in the spacer region, whereas lox sites with identical spacer regions can be recombined efficiently. Recombination using heterospecific lox sites is termed "recombinase mediated cassette exchange (RMCE)" , in which one chromosomally preinserted DNA cassette flanked by two different heterospecific lox sites is exchanged for another cassette on a targeting plasmid flanked by the same kind of heterospecific lox sites. To date, lox511 , lox2272 , and lox5171  have been successfully used in embryonic stem (ES) cells.
The other class is the left element/right element (LE/RE) mutant strategy using LE mutant lox carrying mutations in the left-inverted repeat region and RE mutant lox carrying mutations in the right-inverted repeat region . Recombination between an LE mutant lox and an RE mutant lox results in the generation of a double mutant lox site having mutations in both ends and a wild-type loxP site. The double mutant lox site is not an effective substrate for Cre recombinase; therefore, the recombination reaction proceeds exclusively in one direction (Figure 1b). We have previously demonstrated successful integrative recombination using lox71 (an LE mutant) and lox66 (an RE mutant) in ES cells . Moreover, two other groups have used lox71/66 to induce unidirectional Cre-mediated inversion [15, 16].
Although the integrative recombination efficiency using lox 71/66 is lower than RMCE efficiency using loxP and lox2272 , the advantage of the LE/RE mutant lox strategy is its simplicity. Only one LE or RE lox site is required as a target for integrative recombination. Recently, Thomson et al. performed mutational analysis of LE/RE mutant lox sites using Escherichia coli and identified two novel LE/RE mutant lox, loxJT15 and loxJTZ17, which showed approximately 1500-fold higher integration rates than lox71 and lox66 . If these novel mutant lox sites could also improve integration efficiency in ES cells, they would be useful tools for Cre-mediated integration in mammalian genomes.
One application of the Cre/mutant lox integration system is gene trapping in ES cells. Our group, as well as two other groups (Database for Exchangeable Gene Trap Clones, Sanger Institute Gene Trap Resources and Bay Genomics), have constructed gene trap vectors incorporating a lox71 site and have generated over 20,000 gene trap cell lines, which are available to the academic community through the International Gene Trap Consortium database http://www.genetrap.org/index.html. With these trap clones, any DNA of interest can be inserted into a lox71 site and expressed under the control of the trapped gene promoter . Therefore, in this study, we focused on screening for efficient RE mutant lox possessing better recombination efficiency with the lox71 site. Thomson et al. reported that the recombination efficiency between loxJTZ17 and lox71 was 10 times higher than that between lox66 and lox71. Although the 10-fold promoting effect is less than the 1500-fold effect obtained with loxJT15 and loxJTZ17, this improvement is considered sufficient because the recombination efficiency of lox66 and lox71 in ES cells is 2-16% . Here, we used six RE mutant lox sites, including lox66, loxJTZ, and four newly synthesized RE mutant lox sites(loxKR1-4), and compared both their recombination efficiency and the stability of recombined products in ES cells.
Results and Discussion
Mutant RE lox sites
We synthesized four new RE lox sites (loxKR1-4) as well as loxJTZ17 (Figure 1c). LoxKR1 had four nucleotide mutations; of these, three mutated nucleotides were the same as the mutations in lox66, and one nucleotide was the same as the mutation in loxJTZ17. LoxKR2-4 were designed to have three mutations, as did loxJTZ17, but in different positions.
Assessment of integrative recombination efficiency
Site-specific integration frequencies
Frequency of site-specific integration
13.4 ± 4.3
18.0 ± 5.4
13.9 ± 2.5
19.2 ± 6.9
18.0 ± 2.4
17.5 ± 1.2
11.3 ± 0.6
17.2 ± 2.2
13.3 ± 1.5
22.7 ± 3.3
17.3 ± 2.4
17.5 ± 3.7
14.2 ± 2.9
13.3 ± 3.1
10.5 ± 1.8
18.0 ± 5.7
11.9 ± 3.4
12.9 ± 1.1
13.8 ± 1.3
15.8 ± 2.6
14.4 ± 2.3
18.9 ± 3.6
13.9 ± 4.5
16.9 ± 2.7
Assessment of stability of double mutant lox against Cre expression
Why did loxJTZ17 not increase the frequency of site-specific integration in ES cells? The major difference between the report by Thomson et al.  and the present study is the differing species of host cells: prokaryotic cells and eukaryotic cells, respectively. Prokaryotic cells are much smaller than eukaryotic cells. In addition, their genomic DNA is not separated by a nuclear membrane and has a more open structure than eukaryotic cells. Therefore, in prokaryotic cells, Cre proteins and lox sites should exist in much higher concentrations and meet and bind more frequently than in eukaryotic cells. The efficiency of integrative recombination in the LE/RE mutant lox system depends on the in-affinity of the LE+RE double mutant lox site to Cre protein. In this environment of prokaryotic cells, Cre proteins may be able to act on double mutant lox sites with incomplete levels of in-affinity for Cre protein. In our assay using ES cells and transient expression of the cre gene, the chance of a collision between Cre proteins and the lox site was much lower than in prokaryotic cells, and the double mutant lox site was exposed to Cre proteins for only a limited time. Therefore, Cre proteins may have disappeared before they could recombine loxP and LE+RE double mutant lox.
If the ineffectiveness of loxJTZ17 recombination in ES cells is due to the limited chance of a collision between Cre protein and lox sites, prolonged expression of Cre protein should affect the recognition and recombination of LE+RE double mutant lox sites. Therefore, we decided to examine the stability of LE+RE double mutant lox sites in ES cells under the continuous presence of Cre protein.
We first examined the short-term stability of these double mutant lox. Linearized CAG-Cre-IRES-Hyg cassette (pCAGNintCreIH) was introduced into each sub-clone, and Cre-expressing transformants were selected with hygromycin (hyg). Recombination between double mutant lox and loxP results in removal of the puromycin (puro) resistant genes; therefore, recombined cells become puro-sensitive. If recombination occurs soon after introduction of the CAG-Cre-IRES-Hyg cassette, hyg-resistant (hygR) colonies should be puro-sensitive (puroS). In the formation of entire hygR puroS colonies, recombination should occur before the first cell division, or, if formation occurs after the first cell division, recombination should occur in all of the daughter cells. Therefore, in this assay, it is possible to observe recombination within 24-48 h after exposure to Cre protein. To estimate the percentage of such hygR puroS colonies, electroporated cells were divided into two plates. After 5 days of hyg selection, one plate was selected with puro and the other plate was fed with normal medium to obtain the hygR colony number. The ratio of hygR puroS colonies to hygR colonies represents the short-term stability of double mutant lox site.
To estimate the rate of allele excision, band intensities relative to the band derived from the endogenous Pgk gene were measured (Figure 7d, solid and hatched lines), and the percentages of excised alleles were calculated (Figure 7d, gray line). In 71/66-P-Cre clones, the rates of allele excision were 48% (clone No. 1) and 74% (clone No. 4), suggesting that the lox71/66 double mutant is not highly resistant to re-recombination with loxP under continuous exposure to Cre expression. In 71/JTZ-P-Cre and 71/KR3-P-Cre clones, the rates of allele excision were under 21%, meaning that about 80% of double mutant lox sites were not recombined, even under the strong Cre expression forced by the CAG promoter (Figure 7d., middle and below).
Thus, lox71/JTZ17 and lox71/KR3 are highly resistant to the Cre protein, but lox71/66 can be recombined with loxP when the cre gene is expressed strongly and constitutively. Therefore, if we used stable transformants of the cre gene for site-directed integration experiments, lox66 should show a lower efficiency because of its higher rate of re-excision.
In this study, we screened for RE mutant lox sites showing higher recombination efficiency with lox71 using ES cells. Although we could not identify any RE mutant lox with a significantly higher efficiency than lox66, we found that two RE mutant lox, loxJTZ17 and loxKR3, produced more stable (less inactive) double mutant lox with lox71 than did lox66/71. These two mutant RE lox sites would therefore be more suitable than lox66 for Cre-mediated integration or inversion in ES cells.
Plasmids pCAGGS-Cre, pCAGlox71bsr, and plox66NZneo have been described previously [17, 20]. The loxJTZ17 and loxKR1, 2, 3 and 4 sequences (Figure 1c) were synthesized, and the lox66 sequence of plox66neo was replaced by these synthesized lox sequences to produce pJTZNZneo, pKR1NZneo, pKR2NZneo, pKR3NZneo, and pKR4NZneo (RE loxNZneo plasmids). The p66NZPPacP plasmid was constructed by replacing the splice acceptor- enhanced green fluorescent protein (EGFP) cassette of p6SEFPPF  into the LacZ gene fused with the nuclear localization signal (NLS) derived from the SV40 large T gene (NLS-LacZ). The pJTZNZPPacP, pKR1NZPPacP, pKR2NZPPacP, pKR3NZPPacP, and pKR4NZPacP (RE loxNZPacP plasmids) were constructed by replacing the lox66 sequence with the loxJTZ17 and loxKR1, 2, 3, and 4 sequences, respectively. The sequences of all lox sites in these plasmids were confirmed by DNA sequencing.
The Cre-expression vector, pCAGNintCreIH, was assembled from components of pSP73 (Promega, USA), the CAG promoter , the NLS-splice donor (SD)-intron-splice acceptor (SA)-cre cassette , the internal ribosomal entry site (IRES) from the encephalomyocarditis virus (ECMV), the hygromycin-resistance gene, and the polyadenylation signal (pA) from the mouse phosphoglycerate kinase-1 (Pgk) gene.
ES Cell cultures
ES cells were cultured in KSR-GMEM medium consisting of Glasgow Minimum Essential Medium (GMEM) (Sigma, USA) with 1× MEM nonessential amino acids (Gibco Invitrogen, USA), 0.1 mM β-mercaptoethanol, 1 mM sodium pyruvate, 1% fetal bovine serum (FBS; HyClone, Thermo Fisher Scientific Inc., USA), 14% Knockout™ Serum Replacement (KSR; Gibco Invitrogen), and 1100 U/ml leukemia inhibitory factor (LIF; ESGRO, Chemicon, USA). For neutralization of trypsin, FCS-GMEM in which the KSR in KSR-GMEM was replaced with FBS (final concentration, 15% FBS) was used.
ES cell lines (Bs2, Bs17, Bs19, and Bs21) carrying the target lox71 site were established from CGR8  (Gift from Dr. Niwa) by introducing 10 μg of SpeI-digested pCAGlox71bsr plasmid DNA. ES cells (3 × 106 cells/0.8 ml in PBS) were electroporated using a Bio-Rad Gene Pulser (Bio-Rad, USA) set at 200 V and 960 μF and plated into two 10-cm plates. Blasticidin S selection was started after 48 h of electroporation at 4 μg/ml for 7 days, and colonies were picked, expanded, and stocked. Clones with a single copy integration were selected by Southern blotting analysis.
For Cre-mediated integration, ES cells were coelectroporated with 20 μg of RE loxNZneo plasmid and 10 μg of pCAGGS-Cre at 400 V and 250 μF. G418 selection at 600 μg/ml for 7 days was started after 24 h of electroporation. The colonies were then stained with X-gal.
The ES cell line Ttr-KO-41, which carries a lox71-Pgk promoter-neomycin phosphotransferase (neo) gene-loxP-pA cassette in the first exon of the Ttr gene (Ttr neo ), has been described previously . To obtain site-specific integrants of RE loxNZPacP cassette into the Ttr neo allele, Ttr-KO-41 ES cells were coelectroporated with 20 μg of RE loxNZPacP plasmid and 10 μg of pCAGGS-Cre at 400 V and 250 μF. Puromycin selection was started after 48 h at 2 μg/ml for 7 days. Colonies were picked and stocked. For electroporation of pCAGNintCreIH, ES cells were electroporated with 20 μg of XhoI-digested pCAGNintCreIH at 400 V and 250 μF and were fed 150 μg/ml of hygromycin B-containing medium after 24 h of electroporation. Hygromycin B selection was maintained for 5 days; we then changed to puromycin selection at 2 μg/ml or to normal medium for 2 days.
Analyses of DNA and RNA
Cells were lysed with sodium dodecyl sulfate (SDS)/proteinase K, treated with 1:1 (vol/vol) phenol/chloroform, precipitated with ethanol, and dissolved in 10 mM Tris-HCl, pH 7.5/1 mM ethylenediaminetetraacetic acid (TE). Six micrograms of genomic DNA were digested with appropriate restriction enzymes, electrophoresed in a 0.9% agarose gel, and blotted onto a nylon membrane (Roche, Switzerland). Hybridization was performed using a DIG DNA Labeling Kit (Roche). The intensities of the obtained bands were determined using Printgraph AE-6920-MF (ATTO, Japan).
Total RNA was isolated from ES cells using Sepasol (Nakalai, Japan). Ten micrograms of total RNA were electrophoresed through 1.0% agarose-formaldehyde gels and transferred to a positively charged nylon membrane (Roche). Hybridization was performed using a DIG RNA Labeling and Detection Kit (Roche).
The recombination efficiencies and relative number of blue or white colonies were evaluated by nonrepeated measures analysis of variance (ANOVA). Where a significant difference (p < 0.05) was identified, the differences were analyzed further with Student-Newman-Keuls (SNK) tests for multiple comparisons.
We wish to thank Ms. Y. Tsuruta for her technical assistance. This work was supported by KAKENHI (Grant-in-Aid for Scientific Research) in Priority Areas "Integrative Research Toward the Conquest of Cancer" (17012018 to K. Y.) from the Ministry of Education, Culture, Sports, Science and Technology.
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