Predetermined chromosomal deletion encompassing the Nf-1 gene

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Abstract

Complex chromosomal rearrangements (deletions, inversions, translocations) are a hallmark of human tumour cells. Yet, the generation of animal models for gross chromosomal abnormalities still presents a formidable challenge. Here, we describe a versatile procedure for chromosomal engineering that was used to generate an ES cell line with a megabase deletion encompassing the tumour suppressor gene neurofibromatosis-1 (Nf-1) on mouse chromosome 11, which is often deleted in tumours of neural crest origin. Homologous recombination into sites flanking Nf-1 was used to introduce artificial sequences (triple-helix, loxP, vector backbone) that can be employed for in vitro recovery of intervening sequences or the generation of in vivo deletions. This strategy may be developed into a scheme by which large chromosomal regions with precisely defined end points may be excised from mammalian cells and reintroduced after suitable in vitro modification.

Main

Malignant cells often carry chromosomal deletions thought to encompass tumour suppressor genes (Johansson et al., 1993). Such deletions range from small intragenic deletions to loss of large chromosomal regions encompassing thousands of genes. However, using conventional methods of gene inactivation in the mouse, deletions in the megabase range are still difficult to reproduce.

Here, we describe an integrated methodological approach that allows predetermined chromosomal deletions to be achieved in the genome of mouse embryonic stell cells in vivo and in vitro and, at the same time, to recover intervening sequences in a clonable form. As a model, we have used a region on mouse chromosome 11, which contains the neurofibromatosis-1 tumour suppressor gene that is often deleted in tumours derived from neural crest cells (Viskochil et al., 1993).

We have previously described the construction and use of neomycin and hygromycin resistance cassettes containing an artificial triple-helix site and a synthetic lac operator for integration into mammalian genomes (Nehls et al., 1994). This strategy allowed restriction endonuclease cleavage at pre-defined sites in vitro and subsequent affinity purification. However, cloning of the isolated fragment required ligation to a vector backbone prior to transformation into a suitable host. To improve this basic principle, we have subsequently modified one targeting vector by the incorporation of a suitable vector backbone thus replacing a two-fragment ligation with an intramolecular circularization step (data not shown). In a further modification of this procedure described here, loxP sites have been added to the targeting vectors. This eliminates the need for any DNA ligation step since recombination at tandemly arranged loxP sequences catalyzed by cre recombinase produces circular molecules (Hochman et al., 1983). A further significant advantage of this strategy is that deletions can be generated in vivo. Previously, Cre-loxP recombination has been used to generate site-directed chromosomal translocations (Smith et al., 1995; van Deursen et al., 1995) and large deletions (Ramirez-Solis et al., 1995; Li et al., 1996). Our deletion strategy extends these findings by incorporating the facility to recover intervening sequences in clonable form.

We have previously determined the transcriptional orientation of the Nf-1 gene relative to physically linked sequence-tagged sites on chromosome 11 (Nehls et al., 1995); this information simplified the design of the targeted deletion experiment described here. Two consecutive homologous recombination events introduced the desired artificial sequences at markers D11Bhm100 and D11Bhm109, flanking the Nf-1 gene at either end (Figure 1a). The genetic distance between the two STS markers is estimated to be 0.18 cM (Montgomery et al., 1998); the physical distance is not exactly known but is greater than 500 kb (Nehls et al., 1995). After the first targeting event, the artificial sequence upstream (i.e. centromeric) of the Nf-1 gene (Figure 1b) contains a loxP sequence, followed by a tkneo fusion gene and a F′-plasmid based vector backbone, derived from the peloBAC vector (Shizuya et al., 1992). After the second targeting, the telomeric D11Bhm109 sequence (located downstream of the Nf-1 gene) is supplied with a loxP sequence and a hygromycin resistance gene. Because the second targeting event may affect either homolog of chromosome 11, cell lines with targeted integrations on one or two chromosomes, respectively, were subsequently selected by fluorescence in situ hybridization (Figure 2). Cell line D7-2 (Figure 2a) carries both insertions on one chromosome, whereas in F8-1 cells, the integrations occurred on different chromosomes. To achieve the desired intrachromosomal deletion, D7-2 cells were transfected with an expression plasmid encoding a GFP-cre fusion protein (Gagneten et al., 1997).

Figure 1
figure1

Physical maps of the targeted region. (a) Overview of the targeted region with YAC clones covering the Nf-1 locus indicated; circles indicate individual STS markers (pre-fix D11Bhm omitted for clarity) (Nehls et al., 1995). Centromere is to the left, telomere to the right. (b) Structure of targeted centromeric locus (D11Bhm100). The tkneo cassette and the BAC vector are inserted into a genomic Kpnl site (K, underlined); S1, SacI; E, EcoRI. The locations of the loxP and triple-helix-lacOs (THS) sequences are indicated. (c) Structure of the targeted telomeric locus (D11Bhm109). Insertion of loxP sequence and hyg cassette occurs at an EcoRI site (underlined). The wild-type SacI fragment containing by the D11Bhm109 sequence-tagged site is 3.1 kb in length and can be detected using a probe (horizontal bar) located telomeric to the targeted insertion; after insertion of loxP and hyg cassettes, its size increases to 5 kb (c.f. Figure 3c). After cre-mediated deletion of genomic DNA between the two loxP sites, a SacI fragment of 6.2 kb is detectable, as the genomic SacI site next to the loxP sequence (open arrow) is replaced by the SacI site upstream of the centromeric insertion (marked with open arrow in b). Methods: procedures for electroporation, selection and analysis of targeted recombinants have been described (Nehls et al., 1996). Details of targeting constructs are available from the authors upon request

Figure 2
figure2

Chromosomal localization of tkneo (yellow) and hyg (red) sequences in ES lines D7-2 (a) and F8-1 (b). Hybridization signals co-localize (arrow) in D7-2 cells and are on two different chromosomes in F8-1 cells. Methods: metaphase spreads were hybridized (Lichter et al., 1990) with a mixture of haptenized tkneo and hyg probes. The digoxigenin-labelled hyg probe and the biotinylated tkneo probe were detected by incubation with anti-digoxigenin conjugated to rhodamine (Boehringer Mannheim) or with avidin conjugated to fluorescein isothyiocyanate (FITC) (Vector Laboratories), respectively. Chromosomes were counterstained with 4,6-diamino-2-phenylinodole-dihydrochloride (DAPI). Digitized images of emitted DAPI, rhodamine and FITC fluorescence were recorded separately by using a CCD camera (Photometrics), electronically overlayed, aligned and pseudocolorized

After 48 h, 2.5% of transfected cells were found to express GFP and could be purified by fluorescence activated cell sorting. Individual sub-clones were analysed after a further 7 days.

Cre recombinase activity results in intrachromosomal rearrangement and the deletion of genomic DNA encompassing the Nf-1 gene; the excised fragment is contained in an extrachromosomal episome carrying a F′-plasmid based origin of replication (Figure 3a). Because this episome lacks a centromere and possibly also a eukaryotic origin of replication, it will be lost during subsequent cell divisions. The resulting sub-clones will, therefore, be hygromycin resistant (as the hyg gene is located telomeric to the loxP site) but neomycin sensitive (as the tkneo gene is contained in the episome). Fourteen hygRneoS sub-clones of D7-2 cells derived after transient cre expression were tested for the deletion event by the PCR strategy shown in Figure 3a. Ten clones (for an example, see Figure 3b) contained the expected deletion as detected by primer combination 156 and 157; the parental D7-2 cell line is negative in this assay, because the relevant sequences are at least 500 kb apart. An amplimer diagnostic for the deleted fragment (primer combination 160 and 30) is not detectable, consistent with the notion that the episome is no longer present in these cells. This result is confirmed by Southern filter hybridization analysis (Figure 3c) using a tkneo probe and a probe derived from the telomeric end of the SacI fragment carrying the targeted insertion loxP-hyg cassette (c.f. Figure 1c). Although the in vivo generated BACs appear to be unstable in ES cells, it is conceivable that the provision of a suitable eukaryotic origin of replication may lead to a stable maintenance of the excised fragments in episomal form.

Figure 3
figure3

Structure of chromosomes before and after cre-mediated rearrangements in cell line D7-2. (a) Schematic representation of the two chromosome 11 homologs with positions and numbers of relevant primers (arrows) indicated. LoxP sites are indicated as open triangles. The location of tkneo, BAC and hyg cassettes are indicated (c.f. Figure 1b,c). Primer TS30, 5′-cgactgcatctgcgtgttcg; TS156, 5′-atctccagcagtgtggtgtcagc; TS157, 5′-tcgatgcgacgcaatcgtcc; TS160, 5′-tgccaagacatcctatcatgg; (b) Analysis of D7-2 cells after cre-mediated chromosomal rearrangements. PCR analyses of genomic DNAs from the parental cell line and one representative subclone (D7-2c) are shown in the left panel. Results of an in vitro excision reaction in the absence (−) or presence (+) of cre-recombinase are shown in the right panel. Primer combination 156/30 detects the intact centromeric insertion (amplimer size 321 bp); 160/157 detects the intact telomeric insertion (amplimer size 575 bp); 156/157 detects the deletion event (amplimer size 559 bp); 160/30 detects the episome (amplimer size 337 bp). (c) Southern filter hybridization of SacI-digested DNA of the D7-2 parental cell line and one representative subclone is shown for a telomeric genomic probe (c.f. Figure 1c) or a tkneo gene probe. The SacI fragments expected to hybridize with the telomeric probe (see legend to Figure 1) are indicated; wild-type locus (wt), targeted locus (t), and rearranged locus (d). The tkneo probe detects the expected 2.7 and 11 kb fragments only in the parental D7-2 cell line, since the subclone has lost the circular excision product. Methods: the His-tag/Cre-fusion proteins consist of the Cre sequence extended either at the N-terminus (mrgshhhhhhgSNLLTVH...DGD) or the C-terminus (mrgsSNL...DGDrshhhhhh) [His-tag underlined; amino acids of Cre in upper case]. The fusion proteins were isolated from bacterial extracts by affinity purification via their His-tags using Ni-resin (Qiagen). The final elution buffer consists of 50 mM Na-phosphate pH 7.8, 300 mM NaCl, 20% glycerol, 250 mM imidazole. To remove minor contaminants remaining after His-affinity purification, gel filtration was performed with Sephadex G-75, run with elution buffer without imidazole. The final preparation was >95% pure as determined by SDS – PAGE. Agarose plugs were prepared using 0.5% low-melting point agarose in PBS. Each plug contained about 5×106 cells. Cell lysis and proteinase K treatment were performed according to standard procedures, and the plugs were finally equilibrated in 0.5 M EDTA for storage at 4°C. For in vitro recombination, plugs (100 μl) were equilibrated in an excess of recombination buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 30 mM NaCl, 100 μg/ml BSA) at 4°C with at least five buffer changes. Thereafter, 100 μl of buffer with cre enzyme at 5 μg/ml were added and the plugs were incubated for further 3.5 h at 4°C. To induce recombination, samples were shifted to 37°C for 1.5 h. Reactions were stopped by incubation at 70°C and the melted agarose was digested with GELase (Epicentre Technologies) according to manufacturer's instructions. The enzyme was inactivated at 65°C and the resulting solution purified using microconcentrators (Microcon-100). DNA was stored in 10 mM Tris-Cl, 1 mM EDTA, pH 7.5 at 4°C. ES cells were cultured in DMEM supplemented with 15% FCS and 1000 U/ml ESGRO (Gibco BRL) on gelatinized plates. The day before transfection with the GFPcre expression vector pBS500 (Gagneten et al., 1997), ES cells were split and seeded at about 4×105 cells per 3.5 cm plate. The next day, cells were transfected with 1 μg pBS500 complexed with 3 μl Fugene 6 Transfection Reagent (Boehringer Mannheim) in 2 ml medium according to the manufacturer's protocol. About 40 – 42 h after transfection the cells were trypsinized and resuspended in PBS-3% FCS for flow cytometric analysis and cell sorting with a FACStarplus cytometer (Becton Dickinson). GFP expressing cells were detected by excitation with an Argon laser (488 nm) and a 525 nm bandpass emission filter. As a control, non-transfected ES-cells were used. Cells expressing high levels of GFP (about 2.5%) were gated. Sorted cells were replated and single colonies picked for DNA preparation. Southern blot analysis was performed using Hybond N membranes (Amersham) and ExpressHyb Hybridization Solution (Clontech) with 32P labelled probes. Filters were first probed with the 500 bp NsiI fragment indicated in Figure 1c; the probe was then removed from the filter by boiling in 0.1% (w/v) SDS and rehybridized with the 3.6 kb tkneo fragment shown in Figure 1b

The intrachromosomal deletion event apparently occurs with such high frequency (about 70% in the case above) that enrichment for cre-expressing cells suffices to obtain sub-clones with the desired genomic modification, at least in case of the Nf-1 region. In less favourable situations, our experimental design allows for the enrichment of cell clones containing the targeted deletion by counter-selection with ganciclovir (as the tkneo fusion gene is contained within the unstable episome). We have also attempted to measure the frequency of cre-mediated chromosomal translocation in F8-1 cells. However, PCR analysis of a pool of 200 clones did not show unambiguous evidence for the desired inter-chromosomal rearrangement (data not shown). This is in agreement with the findings of other studies, which have reported translocation frequencies of less than 1 in 1000 (van Deursen et al., 1995; Smith et al., 1995). It is unclear at present, why the intra-chromosomal rearrangement occurs at a much higher frequency than the inter-chromosomal event.

In some instances, the in vitro recovery of the deleted DNA fragment for cloning purposes may be desirable. Our previously described strategy encompassed the facility for triple-helix-mediated chromosomal cleavage as both selection markers carry the previously described optimized THS-sequence in addition to an artificial lac operator sequence for subsequent affinity purification of DNA (Nehls et al., 1994). We, therefore, wished to ascertain whether the in vivo excision by cre recombinase of the Nf-1 region was also possible in vitro. Because of the size of the resulting episome, cre-mediated excision should take place in conditions minimizing shear forces. To this end, intact chromosomal DNA was prepared in agarose plugs and treated with a highly purified bacterial His-tag-cre fusion protein. DNA reaction products were purified and subjected to PCR analysis. The results shown in Figure 3b (right panel) indicate that the deletion product is readily detectable. However, in contrast to the in vivo situation, the assay designed to detect the resulting episome (primer combination 160/30) is also positive, indicating the presence of the Nf-1 BAC clone.

We have chosen a single-copy bacterial origin of replication for our present studies, as fragments cloned in such vectors have been shown to be stable in suitable E. coli hosts. This is often not the case with yeast artificial chromosomes where mammalian DNA tends to become rearranged during propagation in yeast cells. However, with the advent of rapid strategies for homologous recombination in bacteria (Kempkes et al., 1995; Zhang et al., 1998), the in vitro manipulation of such fragments before reintroduction into mammalian cells (previously a major advantage of the yeast system) has become a feasible undertaking. Future work, however, will have to address conditions by which in vitro generated BACs can be efficiently transferred into bacteria despite a large excess of genomic DNA; encouraging results have already been reported (Sheng et al., 1995; Zhu and Dean, 1999).

Because the targeted deletion leaves a single loxP site of known orientation in the chromosome, the experimental strategy exemplified here may ultimately be developed into a scheme to replace large chromosomal regions with precisely defined endpoints from one source by homologous regions from another source in a manner that preserves chromosomal context and orientation.

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Acknowledgements

We thank Drs M Nehls and M Messerle for the initial isolation and characterization of genomic clones containing markers D11Bhm100 and D11Bhm109, Dr B Sauer for provision of the GFP-cre expression plasmid, and M Sator-Schmitt and H Kohler for excellent technical assistance. This work was initiated at the German Cancer Research Center, Heidelberg, and completed at the Max-Planck-Institute for Immunobiology, Freiburg. Financial support from the Deutsche Forschungsgemeinschaft is gratefully acknowledged.

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Correspondence to T Boehm.

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Schlake, T., Schupp, I., Kutsche, K. et al. Predetermined chromosomal deletion encompassing the Nf-1 gene. Oncogene 18, 6078–6082 (1999) doi:10.1038/sj.onc.1203021

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Keywords

  • neurofibromatosis-1
  • cre recombinase
  • chromosome engineering

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