An efficient method to successively introduce transgenes into a given genomic locus in the mouse
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Expression of transgenes in mice requires transcriptional regulatory elements that direct expression in a chosen cell type. Unfortunately, the availability of well-characterized promoters that direct bona-fide expression of transgenes in transgenic mice is limited. Here we described a method that allows highly efficient targeting of transgenes to a preselected locus in ES cells.
A pgk-LoxP-Neo cassette was introduced into a desired genomic locus by homologous recombination in ES cells. The pgk promoter was then removed from the targeted ES cells by Cre recombinase thereby restoring the ES cells' sensitivity to G418. We demonstrated that transgenes could be efficiently introduced into this genomic locus by reconstituting a functional Neo gene.
This approach is simple and extremely efficient in facilitating the introduction of single-copy transgenes into defined genomic loci. The availability of such an approach greatly enhances the ease of using endogenous regulatory elements to control transgene expression and, in turn, expands the repertoire of elements available for transgene expression.
KeywordsLeukemia Inhibitory Factor Transcriptional Regulatory Element G418 Resistant Coloni General Target Vector Endogenous Regulatory Element
phosphoglycerate kinase promoter
Herpes simplex virus thymidine kinase
hepatocyte nuclear factor
leukemia inhibitory factor
The study of gene function has been greatly advanced through the use of transgenic mice. Over-expression and ectopic expression of transgenes, as well as the expression of genetic variants, has facilitated the deciphering of complex cellular processes as well as the generation of animal models of disease. More recently, transgenic mice have also been used to express Cre recombinase to mediate the conditional deletion of loxP-flanked DNA sequences in specific cell types in vivo. Traditional methods for generating transgenic mice require the availability of well characterized transcriptional regulatory elements, promoters or enhancers, that direct transgene expression in specific cell types. For example, Surfactant protein C regulatory elements have been used extensively to drive high level expression of transgenes in the lung [1,2,3,4]. Similarly, Transthyretin regulatory elements confer expression of transgenes in the liver [5, 6]. Unfortunately, the number well-characterized enhancers/promoters available for transgene expression is limited. This is due to the fact that, although in situ hybridization and RNA blotting techniques can rapidly characterize the expression patterns of genes, the identification of regulatory elements that control cell type-specific expression in animals is extremely laborious. An additional complication associated with traditional transgenic approaches is that the number and genomic location of transgene integration events is random. This can have a significant impact on both the level and site of transgene expression. These problems can be circumvented, however, by utilizing endogenous regulatory elements. This can be achieved by introducing transgenes into defined genomic loci through homologous recombination in ES cells ("knock-in") . While this approach requires knowledge of the gene expression pattern it does not require any analysis of gene regulatory elements. Such a "knock-in" approach would, in effect, significantly increase the repertoire of regulatory elements available to drive transgene expression. Moreover, it ensures that a single copy of the transgene is introduced into a defined genomic location. The introduction of LacZ into specific genes during the generation of knockout mice has demonstrated that such an approach can be successful [8, 9]. The downside, however, is that every individual transgene an investigator wishes to express must be independently targeted to the chosen genomic locus. This can be tedious, especially if the rate of recombination at the chosen genomic locus is low. Here, we describe a simple method that allows transgenes targeted to a marked genomic locus to be selected with extremely high efficiency in ES cells. This approach is also appealing because it relies on reconstitution of active neomycin phosphotransferase activity, and so correctly targeted ES cell clones can be simply selected by growth in G418.
We predicted that transgenes inserted into the Hnf3α locus would be expressed throughout the endoderm of the developing gut. To determine if this was true we generated embryos from ES cells containing LacZ at the Hnf3α locus (Hnf3αLacZ ES cells) and stained for expression of β-galactosidase. Mouse embryos derived solely from ES cells were generated by tetraploid aggregation as described previously [18,19,20]. A total of 10 embryos were produced and all showed the same pattern of β-galactosidase expression. Fig 4b shows β-galactosidase staining in Hnf3αLacZ embryos at 10.5 days of embryonic development. As expected, β-galactosidase was expressed throughout the gut, liver and at particularly high levels in the developing stomach. Endogenous Hnf3α is also expressed in the floorplate of the neural tube as well as the notochord. However, expression of β-galactosidase in Hnf3αLacZ transgenic embryos was undetectable in these tissues. In generating the Hnf3αLacZ targeting vector we deleted all genomic sequences lying 3' to the first intron of the Hnf3α gene. It is likely that this intron or other untranslated sequences contain regulatory elements that specifically direct expression of Hnf3αLacZ in the floorplate of the neural tube and notochord . Indeed when LacZ is introduced into exon 2 in ES cells, leaving the first intron intact, expression of β-galactosidase is readily detectable in the neural-tube floorplate and notochord .
Discussion and conclusions
We have described a method that allows transgenes to be easily targeted to a defined locus in the mouse genome in ES cells. Although this is a multiple-step procedure, once the chosen locus has been targeted using homologous recombination, subsequent manipulations are extremely efficient. Indeed in the final step, where the transgene of interest is targeted to the desired locus, we found that 100% of G418 resistant ES cell colonies were correctly targeted. Transgenic mice and embryos can be generated from these ES cells by standard injection into blastocysts and subsequent breeding of the resulting chimeric mice. It is worth noting, however, that if germline transmission is the aim of the experiment it is important to ensure that the "Δpgk-Neo" cells generated in step 2 are germline competent. This is important because each round of clonal selection increases the likelihood of losing germline competency of the ES cells.
The targeting of transgenes into a given locus has a number of advantages over traditional methods of generating transgenic mice. Traditionally, transgenic mice are produced by injection of DNA into the male pronucleus of fertilized mouse eggs. A variable number of copies of the transgene are then integrated into the mouse genome at random locations. The position and number of transgene copies can have a profound effect on their expression. In some cases expression is repressed or, in contrast, undesirably activated at ectopic sites. When transgenes are targeted to a known genomic locus such variation is avoided and expression of the transgene is significantly more predictable . Such control over the site and level of expression is important because variations could have unpredictable impacts on the phenotype presented by the transgenic mice.
Introduction of single copy transgenes into the hydroxyphosphoribosyl transferase (HPRT) locus has been previously described by Bronson et al. This approach successfully overcomes the problems associated with the integration of variable copy numbers of transgenes into random genomic locations. However, it is only suitable for introduction of transgenes to the HPRT locus and, in addition, requires the availability of HPRT-negative ES cell lines. Cre-mediated targeting of single copy transgenes to specific genomic sites that have previously been marked by loxP elements has also been described both in somatic cells and more recently in ES cells using a double LoxP targeting strategy [22, 23]. Although this approach also results in efficient targeting to a defined genomic locus it relies on co-transfection of a Cre expression plasmid along with a loxP-targeting vector that carries the transgene. Hardouin et al also recently described an elegant approach to introduce transgenes to loci identified by gene trapping. Here integration of the transgene at the gene-trap locus was again mediated by Cre recombinase and translation of the transgene was facilitated by an internal ribosomal entry site (IRES) . In contrast, our approach relies simply on the reconstitution of resistance to G418 and can be used to target transgenes to any genomic locus.
Transgenic mice are often used to examine gene function through ectopic expression studies. This requires the availability of characterized transcriptional regulatory elements that are capable of expressing transgenes in the tissue of choice. In addition, for developmental studies, the transcriptional regulatory elements have to ensure transgene expression during the correct developmental time frame. Although the expression patterns of many genes have been described in detail, characterization of promoter and enhancer elements that control this expression is much more limited. This is partly due to the fact that complex expression patterns often utilize genomic regulatory sequences that are positioned many kilobases away from the gene making them difficult to identify. However, the introduction of transgenes into specific loci allows the utilization of intact endogenous transcription regulatory sequences, which increases the likelihood that the transgene will be expressed in the expected fashion.
Using targeted ES cells also provides the potential of expressing lethal transgenes. This may be important for examining the effects of expressing gain-of-function or dominant-negative alleles of a gene product. This is difficult using a conventional approach because of the need to establish founder mice expressing the transgene. However, if a line of "transgenic" ES cells can be established then it is possible to generate clonal embryos directly from these ES cells by aggregating them with tetraploid embryos. Indeed, here we have used this approach to establish that the introduction of a LacZ transgene into the Hnf3α locus facilitates transgene expression throughout the developing gut (Fig 4).
In sum, we have described a method that facilitates the efficient introduction of transgenes into predefined loci in the mouse genome. By selecting appropriate sequences for homologous recombination this approach can be tailored toward any specific genomic locus. We, therefore, believe that this approach expands the repertoire of tools available for genetic manipulation in the mouse and will enhance our ability to address gene function in mammals.
Materials and Methods
In the following cloning steps "blunt" infers that the cohesive ends of a DNA fragment, cut by restriction endonucleases, were repaired by the Klenow fragment of DNA PolI in the presence of deoxyribonucleotides.
Hnf3α targeting vector(p3αloxPNeo-TK)
A 900 bp Xho1/Xba1 fragment of Hnf3α genomic DNA that included exon 1 and a portion of intron 1 was used as the 5' arm of homology. The 3' arm of homology was cloned as a 4.5 kb Xho1 fragment of Hnf3α genomic DNA. A Xho1/HindIII (blunt) cassette containing the Tn5 neomycin phosphotransferase (Neo)gene, that could confer resistance to G418 and whose expression in mammalian cells was directed by a phosphoglycerate kinase-1 (pgk) promoter, was introduced into an XbaI (blunt) site between the Hnf3α genomic sequences [16, 25]. The pgk promoter was flanked by loxP elements so that it could be deleted by the action of Cre recombinase. The herpes simplex virus thymidine kinase (hsv-TK) gene, whose expression was also regulated by the pgk promoter, was introduced adjacent to the Hnf3α 5' arm of homology to provide negative selection in the presence of gancylovir.
Transgene targeting vector (p3αΔNeo)
The transgene-targeting vector used in these experiments was generated in multiple steps (Fig 2). A NotI/RsrII (blunt)1.4 kb cassette containing the 5' end of Neo coding sequence accompanied by the pgk promoter with flanking loxP elements was introduced into the SphI site (blunt) of pNEB193 (New England Biolabs). Deletion of the 49 c-terminal codons of the Neo gene disrupted its ability to encode resistance to G418 (data not shown) . This sub-fragment of Neo provided the 3' arm of homology in the targeting vector. A 530 bp EcoRI fragment (blunt) containing an intron from the mouse protamine gene as well as poly(A) addition sequences was isolated from the plasmid pLacF and cloned into the PmeI site (blunt) of the preceding plasmid . Finally the 5' arm of homology was provided by a 4.5 kb Xho I fragment of Hnf3α genomic DNA that was introduced into a unique Pac1 site (blunt) in the preceding plasmid. Importantly, this fragment extended from 5' genomic DNA into sequences encoding the untranslated 5' end of Hnf3α mRNA. This cloning strategy left a unique Sal I site into which coding sequences of transgenes could be introduced. A NotI (blunt) 3.6 kb lacZ fragment from pCMV-βgal (Clontech) was ligated into this SalI (blunt) site to generate a targeting vector that could be used to introduce lacZ into the "marked" Hnf3α locus.
Culture and selection of embryonic stem cell lines
All ES cell lines were cultured on mitotically inactivated primary embryonic fibroblasts in ES cell medium supplemented with recombinant leukemia inhibitory factor (LIF) as described elsewhere . Gene targeting was carried out using 100 μg of linear targeting plasmid. This was introduced into 2.5 × 108 ES cells by electroporation at 250 volts/500μf/resistance 8 using a BTX ECM600 electroporation system. Cells were plated on thirteen 10 cm2 tissue culture dishes and grown for two days in ES cell medium supplemented with LIF. Cells containing Neo were selected by supplementing the ES cell medium with 300 μg/ml Geneticin (G418,Gibco-BRL) and negative selection against Hsv-tk gene expression was achieved by including 2 μM gancyclovir (Roche). Recombination between loxP elements in ES cells was mediated by introducing a Cre expression plasmid, pHDMCCre8 (provided by Dr. Klaus Kaestner). 100 μg of pHDMCCre8 was introduced into 5 × 107 ES cells by electroporation using 400 v/500μf/resistance setting 8. 1/100,000 of the total electroporated cell population was plated per 10 cm2 tissue culture dish in ES cell medium supplemented with LIF and grown until individual colonies could be collected.
Tetraploid aggregation and β-galactosidase staining
Embryos were generated from ES cells by aggregating them with 4-cell stage embryos made tetraploid by electrofusion, as described previously [19, 20, 27]. Aggregates that formed blastocysts after overnight culture were allowed to continue their development in utero by transferring them to a pseudopregnant surrogate mother. Embryos were stained for expression of β-galactosidase using standard techniques .
We would like to thank Dr. Paula Traktman for critical reading of the manuscript and Dr. Klaus Kaestner for generously supplying the Cre expression vector and for useful advice. This work was supported by NIH R01 DK55743 to S.A.D.
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