We have combined the powers of RMCE and regulated gene expression into a single highly efficient system that should facilitate structure-function studies, enable production of biopharmaceuticals, or provide a means of tracking cell proliferation in real time. The Cre/LoxP recombination system is preferred for site-specific recombination because it exhibits higher efficiency of recombination over the Flp/FRT system in specific cell types such as mouse embryonic stem cells and primary cells (28
). However, the system we describe could as easily be implemented using engineered Flp recombination target sequences designed not to recombine with each other. We first engineered a new LoxP site to minimize intrachromosomal exchanges that generate deletions, and to maximize exchanges between the chromosomal and plasmid donor LoxP targets. To minimize the need for drug selection markers, we introduced into the chromosomal target a HyTK fusion gene. This enables positive selection to identify clones containing the LoxP target, and then counterselection using gancyclovir to select for loss of the chromosomal target and concomitant replacement with the donor plasmid. This is especially valuable for situations in which introduction of extraneous transcriptional control sequences needs to be avoided. We also used an optimized Tet-On system to enable robust controlled expression of the donated sequences. This should allow for analyses of phenotypic effects of the introduced gene when it is expressed at normal, sub- or supraphysiologic levels.
We tested the system by first analyzing the stringency of recombination with the newly characterized L3 LoxP site. A pair of heterospecific recombination sites that do not recombine with each other is necessary for efficient RMCE (6
). When used with the original LoxP site (L2), the first LoxP variant (LoxP511 or L1) that was generated greatly enhanced the frequencies of exogenous gene integration when coupled with positive selection (12
). However, using negative selection as a means to enrich for recombinants, the efficiencies were markedly different (1
). Deletion of intervening sequences without replacement occurred when L2 and L1 were placed as direct repeats, suggesting interaction between L1 and L2 (6
). When placed as inverted repeats, inversion of the intervening sequence resulted in an equal proportion of clones in either direction (1
). This is not desirable, especially when directionality of the integrant is important. We solved the problem of recombination between L2 and L1 by generating a new LoxP site, LoxP257 (L3), which was designed based on findings from in vitro
recombination assays (5
). L3 differs from L2 by three bases within the 8 bp spacer sequence. When used in combination with L2 and L3 generated recombinants at high frequency, suggesting the feasibility of using L3 and L2 for RMCE. The finding was consistent with a previous study showing that a modified LoxP sequence (LoxP2272) with two base changes from L2 also enabled high efficiency exchange (6
). Other than having a high inter-molecular recombination efficiency, the probability of selecting a recombinant clone among the ganciclovir resistant clones is increased when TK expression from the L3HyTK2L parent line is high and stable. Using this criterion for the selection of the L3HyTK2L cell line, we observed high efficiency, accurate RMCE in two different mammalian cell lines at six independent genomic loci. We also observed little size dependence on RMCE frequency or accuracy. The high frequency of correctly recombined clones expedites screening for desirable clones.
We used homologous recombination to introduce L3HyTK2L into several single copy loci, including the intergenic region of the CHO DHFR locus. Our data demonstrate that the DHFR locus was favorable for RMCE and for tightly regulated gene induction. Recombination occurred at very high efficiency (50–100%) and we readily obtained clones exhibiting robust inducible expression (up to 104
-fold by luciferase, and >100-fold by GFP analyses). This locus offers a number of advantages for other studies. For example, it can be readily amplified using selection for methotrexate-resistance (21
). As CHO cells are often used to produce biopharmaceuticals, the combination of amplification and highly regulated expression could provide substantial advantages for reducing production costs, enabling stable maintenance of transgenes, and obtaining very high level expression of potentially toxic proteins. In studies to be presented elsewhere, we found the L2 and L3 sites to be useful for generating genetically engineered ES cells, mouse embryo fibroblasts, and mice with different p53 point mutations or fusion genes with high efficiency (F. Toledo, C. Lee and G.M. Wahl, manuscript in preparation).
Using a strategy similar to that employed for the delivery of Flp/FRT tagged sites into the genome of mammalian cells (25
), we integrated the LoxP sites at single copy into random positions in the genome of HeLa and CHO cells by retroviral delivery (). An advantage of the retroviral system is that it ensures delivery of the entire transgene at high efficiency and at single copy () into the genome. Active transcription sites are the preferred retroviral integration sites (33
). Consistent with this, we were able to select retroviral integrations at high efficiency, an advantage over gene targeting by homologous recombination.
An important advantage of RMCE approach that is valuable for structure-function studies and production of recombinant proteins of pharmaceutical value is the ability to repeatedly obtain clones exhibiting predictable and uniform gene expression at high frequencies (1
). We combined that with the improved version of the Tet-On inducible system to evaluate the reproducibility of gene induction at each locus (11
). By single cell analysis, GFP was uniformly expressed within the population and it was induced in a dose-responsive manner. The improved rtTA (rtTA2s
-M2) demonstrated many superior properties over the original rtTA. Beside the ability to induce graded transgene expression, induction exhibited a greater dynamic range, higher sensitivity to dox and faster induction rate (within 24 h, data not shown) compared to the original rtTA system () (11
). Recombinants derived from the same L3-2L parental line have the reporter gene inserted at the same genomic locus. As expected, the majority of the RMCE clones obtained in one experiment showed similar levels of expression upon induction ( and ).The uniformity in gene expression was also found between clones obtained from independent RMCE experiments (). Hence, clones that reproducibly induce different transgenes can be readily obtained through multiple rounds of RMCE.
Maintaining stable transgene expression is as important as high transgene expression. Since transgene expression may be subjected to silencing, especially when viral promoter is used to drive gene expression (37
) and when expression is not maintained under selective pressure (39
). We measured the stability of expression in HeLa cells after at least 30 population doublings in culture. We observed that the level of inducibility, and the fraction of cells expressing the transgene, was stable over the time period studied when transcription of rtTA and tTR were coupled to that of the neomycin resistance gene by the IRES elements and by maintaining the culture under G418 selection. Hence, the use of IRES to mediate coexpression of multiple genes will gain popularity in both in vitro
and in vivo
The tight and reversible gene expression regulated by the Tet-On system was further demonstrated by switching H2BGFP expression on and then off to monitor cell doubling () as a function of fluorescence units. H2BGFP is incorporated into the chromatin after synthesis, and tracks with nucleosomes that are segregated randomly to daughter cells during each division (27
). Being a stable protein, H2BGFP fluorescence should approximately halve with each division if its expression is prevented. Consistent with this prediction, after H2BGFP expression was turned off for different numbers of population doublings, flow cytometric analysis revealed the expected 2-fold decay per population doubling (). Hence, the inducible expression of H2BGFP can be used to measure population doublings within a defined time frame. For example, as stem cells are expected to divide fewer times than transit amplifying cells, the retention of H2BGFP fluorescence should be able to be used as a marker to localize putative stem cells in situ
and as a means to isolate them by fluorescence based cell sorting (42
In conclusion, the LoxP/Cre recombination system offers reproducible and directional DNA integration into specific loci in the genome at high frequency. The single plasmid Tet-On system, which ensures stable expression of the transactivator and transrepressor, allows reproducible and graded induction of transgenes to a similar level in the RMCE clones derived from single and independent recombination experiments.