The goal of this study was to improve gain-of-function transgenic strategies by targeting cDNAs to a well-defined locus and introducing very efficient in vitro cloning in combination with more broadly applicable ES-cell-based technologies. Here, we have used the ROSA26 locus as a model genomic targeting site to introduce various cDNAs and demonstrate that we could obtain with high targeting efficiency single-copy transgenes conditionally expressed from the endogenous ROSA26 promoter.
The ability of the G4 ES-cell-derived founder males to efficiently transmit the conditional ROSA26-based transgene in the first litter is further expected to decrease the waiting time and number of chimeras that have to be analysed for germline transgene transmission. Such mice can be immediately bred to any Cre line of interest to obtain transgenic animals with modest ROSA26-promoter-driven transgenic expression according to the wide spectrum of constitutive and inducible tissue-specific Cre lines currently available (see http://www.mshri.on.ca/nagy
for a partial and ever-expanding list).
An additional benefit of using the Gateway recombination system in tandem with our ROSA26 locus targeting vectors is that there is an ever-growing list of Gateway-compatible pEntry clones (32
) as well as commercially and publicly available attB
-containing cDNA vectors that can be converted into pEntry clones by performing BP reactions with donor vectors containing the corresponding att
P sites (as an example see the following web resource: http://www.imagenes-bio.de/products/sets_libraries/image
). These vectors and approaches outlined in this manuscript therefore set the stage for the high-throughput systemized creation of conditional gain-of-function ES cell libraries carrying a wide spectrum of ORF subfamilies. To further increase targeting efficiencies that would be needed for the efficient creation of such gain-of-function resources, the Gateway technologies described here should in the future be merged with in vivo
recombinase-based technologies such as [FlpE/Frt
), and ϕC31/att
P systems (34
)] to enable recombinase-mediated cassette exchange (RMCE) in vivo
to the ROSA26 locus (35
) as well as other loci (37
). These types of RMCE-based, ROSA26-based-targeting approaches have already been demonstrated to be extremely efficient in the creation of RNA polymerase III-based gene knockdowns using gene-specific shRNA sequences (35
Using MultiSite Gateway recombination technologies we have inserted the strong CAGG promoter bi-directionally into the ROSA26 locus in both a sense and anti-sense orientation relative to the ROSA26 sense promoter. We have demonstrated that in undifferentiated ES cells, the anti-sense configuration of the pCAGG promoter is optimal for enhanced expression compared to the sense orientation. This result is consistent with previous reports that the minimal cytomegalovirus (CMV) promoter elements also drive higher transgene expression in the anti-sense orientation in undifferentiated ES cells, a result that is thought to be due to ROSA26 sense promoter interference (26
). In our initial analysis of the ROSA26-pCAGG-myc-Mdm4 mice we can clearly see widespread Mdm4 and eGFP protein expression during development (at E14.5) and widespread expression in the adult in Cre-excised tissues. However, we have also documented that there is a significant lack of correlation between pCAGG promoter-driven β-galactosidase expression and X-gal staining in the non-excised situation compared with the broader expression of myc-Mdm4 transgene following Cre-mediated excision of the floxed βgeo STOP cassette. In addition, our initial analysis of Mdm4 protein expression demonstrates that even after Cre-mediated excision of the β-geo STOP cassette we are not getting truly ubiquitous protein expression. For our purposes where we want to mimic the tumour situation in which there is clonal expansion of a small subset of transformed cells, mosaic but high levels of Mdm4 expression may be acceptable or even desirable. However, for studies where ubiquitous expression of the transgene is essential in a given tissue, we propose that the ROSA26-promoter-based expression strategies outlined above are superior when used with well-characterized Cre lines.
Previous attempts to use the CAGG promoter in the generation of the Cre-based bi-reporter Z/AP (39
) and Z/EG (18
) mice have demonstrated similar findings to those which we have documented in this study. Random integration of the pCAGGs-based vectors into undifferentiated ES cells resulted in only around 10% of single-copy random integrants that showed ubiquitous (>90%) X-gal staining in undifferentiated ES cell clones. These results indicate that the CAGGs promoter is quite sensitive to positional integration effects and raise the possibility of promoter interference from the ROSA26 anti-sense-promoter in a tissue-specific manner (40
). As well, the lack of correlation between pre-Cre excision-based expression of β-galactosidase and post-Cre-based expression of reporter constructs (18
) is similar to our observed results where β-galactosidase expression showed more limited and mosaic expression compared with Cre-mediated expression of myc-Mdm4 protein and EGFP. These results may be attributable to elements within the β-geo cassette interfering with pCAGGs-based expression prior to Cre-excision and/or the fact that the bacterial β-galactosidase gene has previously been demonstrated to show mosaic expression particularly in adult tissues (41
) that may be related to sub-optimal codon usage of the prokaryote sequences (18
) or transgene silencing (41
These studies demonstrate that caution should be taken when inserting exogenous promoters into the ROSA26 locus in both sense and anti-sense orientations and it is still not clear if tissue-specific promoter elements can be used to drive ubiquitous and/or cell/tissue-specific expression from this locus on their own during development and in the adult. Recently, several groups have demonstrated the utility of using insulator sequences in transgenic studies (43
). Ciavatta et al
) have demonstrated that copy number-dependent use of insulator sequences at the widely expressed HPRT locus can dramatically protect chicken-β-actin-promoter-based EGFP expression during development and in adult tissues. It is envisioned that similar strategies should in the future be used for the targeting of the pCAGG or other tissue-specific promoters to the ubiquitously expressed ROSA26 locus.