Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation

The analysis of gene function during mammalian embryonic development relies heavily on the use of transgenic mice. However, expression of alleles that result in embryonic lethality cannot be studied using regular transgenic procedures because lines of founder mice cannot be established. Analysis of such alleles in mammals has historically relied upon transient transgenic procedures [1]. In this approach, fertilized eggs are injected with a transgene construct and allowed to develop to a pre-determined embryonic stage in utero before being collected for phenotypic analysis. Using this method a variable number of transgenes integrate into random genomic loci. Unfortunately, the local chromosomal environment can affect expression of the integrated transgene and this can result in unpredictable transgene expression. Moreover, because no founder line is established it is not possible to repeat an analysis with a specific transgenic mouse embryo.

The problem of random transgene integration can be overcome by targeting transgenes to a specific genomic locus by homologous recombination in ES cells [2–4]. A method that allows the introduction of single copy transgenes into the X-linked hprt locus has been described previously by Bronson et al[2]. In this approach single-copy transgenes are introduced 5' of the hprt locus by homologous recombination in ES cells. This is an extremely efficient procedure because targeted clones have reconstituted the hprt gene and are selected on the basis of growth in HAT medium. Following this method, upward of 90% of HAT resistant ES cell clones contain correctly targeted transgenes [2]. The hprt locus is a particularly suitable site for the integration of transgenes because it exists as an X-linked gene present as a single copy in male ES cells. Moreover, the hprt gene is ubiquitously expressed and so provides a favorable chromatin environment for transgene expression. Indeed, it has been demonstrated that the level of expression of transgenes inserted into the hprt locus is directed solely by exogenous transcriptional regulatory elements [2]. Furthermore, mice derived from independent ES cells clones containing a transgene expressed from the same promoter all exhibit comparable levels of expression [2, 4–6].

Although the targeting of constructs to the hprt locus facilitates consistent expression of transgenes it does not overcome the need to generate founder lines of transgenic mice. However, pioneering work by Nagy et al has shown that embryos and mice can be derived directly from R1 ES cells by aggregating them with tetraploid embryos [7, 8]. This suggests that controlled expression of transgenes encoding possible gain-of-function and developmental-lethal alleles could be achieved by generating embryos and/or mice from ES cells containing transgenes inserted into the hprt locus. Unfortunately, the generation of mice and embryos by tetraploid aggregation from available Hprt negative ES cell lines has proven to be inefficient and unreliable (S.A.D. unpublished). In this report, we describe the generation of a new Hprt negative ES cell line that is compatible with the tetraploid aggregation procedure. Moreover, we demonstrate that it facilitates efficient targeting of transgenes to the hprt locus as well as the subsequent generation of clonal transgenic mouse embryos that express the transgene in a promoter-dependent manner.

Nagy et al demonstrated that embryos and mice could be derived solely from R1 ES cells by aggregating them with tetraploid embryos [7, 8]. Although we were able to repeat these data using R1 ES cells we were unable to generate midgestation embryos from E14TG2a-derived strains of hprt negative ES cell lines using the same approach [9–11]. We believed it was unlikely that Hprt itself was required to generate ES cell-derived embryos because hprt knockout mice are viable and fertile [12]. E14TG2a-derived ES cell lines contain a large spontaneous deletion encompassing 55 kb of the hprt locus [13]. Therefore, it seemed possible that sequences within this region were necessary to maintain ES cell pluripotency. However, we believed a more likely scenario was that the available lines contained unrelated genetic or epigenetic alterations that were incompatible with the aggregation procedure. Consistent with this interpretation, Eggan et al have recently shown that viable offspring are more efficiently produced through tetraploid-mediated cloning from outbred ES cell line compared to inbred ES cell lines [14]. We, therefore, believed it likely that aggregation-compatible hprt negative ES cells could be generated by targeted mutation of the hprt gene in R1 ES cells. To test this we generated a targeting vector to replace exon 1 of the mouse hprt gene with a neomycin phosphotransferase (neo) gene that conferred resistance to te pharmacological inhibitor G418 in ES cells (fig 1a). Following transfection of this plasmid into R1 cells 192 clones that were resistant to G418 were collected and grown in the presence of 6-thioguanine to select for loss of Hprt activity. Of the original 192 G418-resistant clones that were harvested only one, named F3, was also resistant to 6-thioguanine and sensitive to growth in HAT medium (fig 3b). The integrity of the targeting event was next confirmed by Southern blot analysis of F3 genomic DNA. As shown in fig 1a the correct integration of Neo at the hprt locus was predicted to delete a BamH1 restriction endonuclease cut-site from intron 1 and replace it with a BamH1 site that is unique to the mutant hprt allele. This restriction fragment polymorphism could be detected using an hprt-specific probe (fig 1a). The wild type BamH1 fragment was predicted to be 7.0 kb based on sequence analysis while the mutant fragment should be 8.3 kb. Fig 1c shows that digestion of wild-type R1 ES cell DNA with BamH1 did indeed generate a 7.0 kb fragment that could be detected with intron 3 probe. In contrast, F3 cells were shown to have replaced this fragment with an 8.3 kb BamH1 fragment thereby confirming the integrity of the targeting event.

For F3 cells to be a suitable vehicle for the generation of transgenic mice they should be competent to generate chimeric mice that can transmit alleles through the germline. To test the "germline potential" of F3 cells we first generated chimeric mice by aggregating F3 ES cells with CD-1 morulae and allowing them to develop to term in utero. The resulting chimeric males were bred with CD-1 females and the genotype of offspring determined by Southern blot analysis of genomic DNA from mouse tails. Fig 1c shows that agouti F1 female mice contained both the mutant (paternal) and wild type (maternal) hprt alleles. In contrast, the agouti F1 male mice contained only a wild-type hprt allele inherited from the maternal X-chromosome as expected. From this we were able to conclude that F3 hprt null ES cells are indeed competent to transmit alleles through the germline and that the resulting offspring were fertile and viable as reported previously [12].

We next addressed whether embryos could be generated solely from F3 Hprt negative ES cells by aggregating these cells with tetraploid CD-1 embryos and allowing them to develop in utero until day 13 of gestation (El 3). Figs 2a,b,c show that F3 cells could indeed give rise to normal embryos (n = 10). All embryos exhibited characteristic features of midgestation stages of development including normal rostral-caudal patterning, beating hearts, normal limb formation including the formation of fingers. F3 cells derive from the agouti (pigmented) SV129/J strain of mice. In contrast, albino CD-1 mice donated the tetraploid morulae used in the aggregation procedure. The finding, shown in figs 2a,b,c, that all the resulting embryos displayed clear retinal pigmentation strongly implied that the embryos were F3 ES cell-derived. We confirmed this by determining the genotype of the embryos by Southern blot analyses of genomic DNA. Fig 2d shows that all embryos contained a restriction fragment polymorphism that was indicative of deletion of hprt exon1. No wild type hprt allele was detected confirming that the embryos were generated entirely from F3 cells. These data demonstrate that the hprt gene is not essential for the generation of embryos from ES cells by tetraploid aggregation and that F3 cells should could provide a suitable vehicle for the direct generation of transgenic mouse embryos.

Previously it has been shown that a targeting vector, pMP8, can correct the loss of Hprt activity in E14TG2a ES cells that lack exons 1 and 2 of the hprt gene [2, 15]. The design of this vector implied that it should also be capable of correcting the hprt exon 1 deficiency in F3 cells and could, therefore, be used to introduce transgenes into the hprt locus by homologous recombination. To test this we generated a modified vector based on pMP8. The practical features of this modification are that it removes the lacZ sequences in the vector backbone and reduces the overall size of the vector, both of which improve subsequent cloning steps. This vector, pMP8NEBΔLacZ, contains a 5' arm of homology consisting of 3.4 kb of mouse 5' hprt genomic DNA flanking a unique Not1 restriction endonuclease cut site. The 3' arm of homology contains 1.8 kb of the human hprt gene that includes exon1 and 2.9 kb of mouse genomic sequence that includes exon 2 (fig 3a) as described previously [15]. Homologous recombination at the modified hprt locus in F3 cells should substitute genomic DNA containing hprt exons 1 and 2 for neo thereby reconstituting a gene encoding active Hprt (fig 3a). Moreover, only cells having undergone a successful homologous recombination event should become HAT resistant and carry the targeted transgene. To assess the efficiency of this approach we attempted to target a LacZ reporter transgene whose expression is controlled by the promoter from the alpha myosin heavy chain (αMHC) gene to the modified hprt locus in F3 cells. We chose the αMHC promoter because others have demonstrated, using traditional transgenic procedures, that it directs high levels of reporter gene expression specifically in cardiac tissue of early mouse embryos [16–18]. Cells were selected for resistance to HAT following electroporation of the αMHC-LacZ transgene-containing plasmid. 16 HAT resistant cells lines were collected and their genotype determined by Southern blot analysis. Fig 3c shows that, in contrast to F3 cells, a probe that specifically recognizes the LacZ transgene identified a 6.7 kb EcoRV fragment in HAT-resistant ES cell lines. Of the 16 HAT-resistant colonies retrieved 15 contained LacZ. Furthermore, fig 3b shows that these cells had become G418 and 6-thioguanine sensitive confirming that the neo gene had indeed been replaced with hprt-coding sequences in the F3 cells. These data confirm that this selection strategy is extremely efficient in identifying ES cell clones containing correctly targeted transgenes [2].

The ultimate use of this procedure is to generate clonal transgenic mouse embryos directly from the modified ES cells. Success in this would provide a system in which to express alleles with potentially lethal phenotypes in the developing embryo. We, therefore, determined whether embryos could be generated from the F3 cells that contained an αMHC-LacZ transgene and whether these embryos appropriately expressed β-galactosidase. E8.5 embryos were generated from three independent αMHC-LacZ cell lines and stained for expression of β-galactosidase. Representative embryos, shown in fig 3d all developed normally and expressed high levels of β-galactosidase in the developing heart, mimicking the expression of endogenous αMHC [16]. Importantly, none of the embryos exhibited ectopic patterns of expression lending further support to the proposal that expression of transgenes introduced to the hprt locus is primarily dependent on the accompanying transcriptional regulatory elements [2].

Here we have described the generation of an ES cell line and a procedure that will allow the phenotypic analyses of transgenic embryos expressing lethal alleles. This procedure is an important addition to the available repertoire of molecular genetic techniques available to developmental biologists because it overcomes several deficiencies associated with alternative transient transgenic approaches normally used to study gain-of-function and dominant-negative alleles. Traditional procedures rely on the generation of transgenic embryos through micro injection of fertilized mouse eggs. Using this approach an unpredictable number of transgene-copies are incorporated at a random location in the recipient genome. This can result in ectopic expression of the transgene because both copy number and the site integration can influence transgene expression.

In the approach described here, a targeting vector that contains exon 1 of the hprt gene along with a specific transgene is used to reconstitute Hprt activity in Hprt negative ES cells. Successful targeting of the transgene to the hprt locus in Hprt negative ES cells can be selected by growing transfected cells in HAT medium. The efficiency of this selection procedure is such that close to all HAT resistant colonies contain a correctly targeted transgene. Moreover, because the transgene is introduced into the hprt locus its expression is dependent upon the inclusion of specific transcriptional regulatory elements and remains relatively unaffected by the local chromatin environment [2]. A major advantage of this targeted transgenic approach compared to traditional transgenics is that transgene expression is predictable so expression at ectopic sites is largely avoided. The availability of F3 Hprt negative ES cells allows transgenic embryos to be derived directly from transgenic ES cells by aggregating the cells with tetraploid embryos and allowing them to develop in utero. Each embryo is, in effect, clonal because it is generated from a single clonal ES cell line. This allows reproducible comparison of the phenotype generated by a given transgene at different developmental stages. Moreover, because the transgene is present as a single copy at a known site of integration it circumvents the need to generate many different transgenic lines from a given construct as is the case when using a transient transgenic procedure.

There are many potential uses for this method of generating transgenic mouse embryos. The most obvious is that it provides a mechanism for generating embryos that express alleles that are predicted, or known, to be detrimental to normal embryonic development, for example dominant-negative or gain-of-function alleles. Indeed, the expression of dominant negative alleles that can potentially disrupt multiple members of a gene family has been used extensively in Xenopus laevis and has, for example, helped elucidate the action of fibroblast growth factors during mesoderm formation [19]. Such alleles are particularly useful when a null allele in the same gene causes a disruptive phenotype prior to the stage of development of interest. Choosing a promoter that expresses the dominant negative allele later in development or in a specific cell type could potentially circumvent any early phenotype associated with a null allele.

Another important use of this procedure could be to identify transcriptional regulatory regions that control expression of specific genes during development. Transgenic mice have been extensively used to identify regulatory elements that control gene expression in vivo[20, 21]. This has, in turn allowed the elucidation of regulatory networks that control cell differentiation during development. In this regard, the use of F3 cells coupled with targeting transgenes to the hprt locus offers two major advantages over traditional transgenic approaches. The first is that it is extremely rapid. Once the targeting construct is in hand it takes approximately four to five weeks to generate transgenic mouse embryos. The second advantage is due to the presence of the transgene at a known and constant integration site. This potentially allows the comparison of levels of reporter gene expression driven by different promoters thereby offering a quantitative in vivo transgenic assay.

In sum, the availability of tetraploid aggregation-competent ES cells that support targeting of transgenes to the hprt locus should facilitate the rapid analysis of transcriptional regulatory elements in vivo as well as the phenotypic characterization of alleles that are incompatible with normal development. We, therefore, believe that F3 cells provide a powerful new tool for molecular geneticists.