Simplified Generation of Targeting Constructs Using ET Recombination Pierre-Olivier Angrand, Nathalie Daigle1,Frankvanderhoeven,Hansr.Schöler1 and A

Simplified generation of targeting constructs using ET recombination Pierre-Olivier Angrand, Nathalie Daigle1,FrankvanderHoeven,HansR.Schöler1 and A. Francis Stewart*

Gene Expression Program and 1Germ Cell Biology Laboratory, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69177 Heidelberg, Germany

Received June 29, 1999; Revised and Accepted July 19, 1999

ABSTRACT large intact DNA molecules regardless of the disposition of ET recombination is a way to engineer DNA in convenient restriction sites, it presents new possibilities for complex engineering. We developed this potential to create, in Escherichia coli using homologous recombination. one step, a knock-out targeting construct for the Ssrp1 gene Here we develop the potential of ET recombination in (5,6) and to insert an IRES-lacZ-selectable gene cassette (7) two ways relevant to complex engineering exercises into a gene coding for a PHD fingers and SET domain-containing such as building gene targeting constructs. First, a protein, called Nsd2 (P.-O.Angrand et al., submitted). targeting construct was made in a single step. Second, ET recombination was used to place two unique restriction sites at precise positions in a large genomic MATERIALS AND METHODS clone. Subsequently a complex targeting construct DNA techniques was created by ligation with a multifunctional cassette. Large-scale plasmid DNA preparation was performed with the Qiagen Plasmid Kit (Qiagen). Restriction endonucleases and INTRODUCTION T4 DNA ligase were purchased from New England Biolabs. Assembling DNA constructs for amplification in Escherichia Plasmids were grown in E.coli strain XL1-blue [F'::Tn10 proA+B+ lacIq ∆(lacZ)M15/recA1 endA1 gyrA96 (Nalr)thi coli is the starting point for many experiments in molecular – + biology. Existing methodologies employing restriction hsdR17 (rk mk ) supE44 relA1 lac]. Complete nucleotide enzymes, PCR and ligation steps are well suited for many tasks sequences and restriction maps of the plasmids used are available but their limitations become evident when complex engineering on the World Web Site http://www.embl-heidelberg.de/ exercises are desired. For example, these limitations often ExternalInfo/stewart/plasmids.html impede the construction of targeting constructs intended for PCR product preparation modifications of vertebrate genomes, in particular, the mouse genome via embryonic stem (ES) cells. All PCR reactions were performed in 50 µl reactions containing The introduction of predetermined modifications into the 5 U Amplitaq DNA polymerase (Perkin Elmer Cetus), 5 ng of mouse germ line via homologous recombination in ES cells is plasmid and 1 pmol of each PCR primers for 30 cycles (94°C fundamental in the application of reverse genetics to mouse 30 s, 55°C1min,72°C 1 min). The zeocin dual transcription biology (1,2). Targeting constructs to manipulate the mouse unit was amplified from ScaI-linearized pSVZeoX1 using the genome often require complex cloning exercises. Minimally primers 5'-GTT CTG TCA AAG GCA GAT GTG ATC this involves positioning two sizeable fragments of mouse CAG GCC ACC GGA GAC GCC ATC TGC ATC TTC genomic DNA (3) either side of a selectable gene. In many GTT TAA ACT CGT TAA TTA AAG GTG GCA CTT TTC cases, these cloning exercises are more complex since inclusion of GGG GAA ATG-3' and 5'-CTTTGCTCTTGAAGCTGT additional elements, such as lacZ or GFP reporter genes, loxP CAC TCA ACT GCC TGG ATG AAG ACT TGG ATG sites or point mutations, are also intended. The use of long ACG ACG AAG CTT AGA CAT GAT AAG ATA CAT TG- segments of genomic DNA requires extensive mapping to 3' where homolgy arms to the exons 6 and 16 of the Ssrp1 gene search for suitable restriction sites for construct design and are in bold. The chloramphenicol resistance gene from DNA fragment assembling by DNA ligations in vitro. pMAK705 (8) was amplified using the primers 5'-CTG TGT We describe here two approaches to simplify complex GAC AAG ACA GGC AGT CTC TAA CTG TGT GAG construction exercises based on complementary applications GGA CCC TGT TGT GGA TTC TAG TTT AAA CCC of ET recombination, a recently described E.coli homologous TGC CCT GAA CCG ACG ACC GGG T-3' and 5'-TAC ACA recombination reaction (4), and conventional DNA metho- GGC TTC ATG GTA AAA CTT TCC ACA CTG ATT dologies. Since ET recombination permits the engineering of TAC CAC ACA ACG TTT CAC CTC GGC GCG CCT

*To whom correspondence should be addressed. Tel: +49 6221 387 562; Fax: +49 6221 387 518; Email: [email protected] Present address: Pierre-Olivier Angrand, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, F-67404 Illkirch Cedex, C. U. de Strasbourg, France e16 Nucleic Acids Research, 1999, Vol. 27, No. 17

CGA ATA AAT ACC TGT GAC GGA AGA TG-3', where the homology arms to Nsd2 are in bold and restriction sites for PmeIandAscI, respectively, underlined. PCR products were purified using the Qiagen PCR Purifica- tionKitandelutedwithwater,followedbydigestionofanyresid- ual template DNA with DpnI. PCR products were then ethanol precipitated and resuspended in water (1 µg/µl). Bacterial transformation Escherichia coli strain JC8679 (9) [recB21, recC22, sbcA23, his-328, thr-1, ara-14, leuB6, ∆(gpt-proA)62, lacY1, tsx-33, glnV44(AS), galK2, rpsL31, kdgK51, xylA5, mtl-1, argE3(Oc), thi-1, Lam–,Rac+,Qsr1+] was transformed by electroporation using a Bio-Rad Gene Pulser set at 2.5 kV, 200 Ω and 25 µF. The transformed cells were suspended in 600 µlL-brothandincubatedfor1hat37°C before plating on L- agar containing zeocin (25 µg/ml) or chloramphenicol (100 µg/ml). Electroporation-competent bacterial cells were made as follows: saturated overnight JC8679 cultures were diluted 50-fold in L-broth, grown to an OD600 of 0.4 and chilled in ice for 30 min. Cells were centrifuged at 5000 r.p.m. for 15 min at –5°C. The pellet was resuspended in ice-cold 10% glycerol and centrifuged again (6000 r.p.m., –5°C, 15 min). This was repeated twice more and the cell pellet was then Figure 1. (A) Schematic representation of the pSVKeoX1, pSVZeoX1 and µ µ pSVBsdX1 plasmids. In pSVKeoX1, the neo resistance gene is placed under suspended in 300 l of ice-cold 10% glycerol. Aliquots (50 l) β ° the control of the -lactamase promoter (blaP) confering neo gene expression were frozen in liquid nitrogen and stored at –80 C. For electro- in E.coli, and under the control of the SV40 early enhancer/promoter region poration, cells were thawed on ice and added to 1 µlmix (SVe) confering neo gene expression in mammalian cells. The neo transcription containing vector DNA (0.5 µg) and PCR product (0.5 µg). unit which is derived from the pBK-CMV vector (Stratagene) is flanked by loxP sites (triangles). pA, thymidine kinase polyadenylation signal. pSVZeoX1 and pSVBsdX1 are derived from pSVKeoX1 by replacement of the neo coding sequence by the ble or bsd coding sequences from pcDNA3.1/ Table 1. Antibiotic resistance conveyed by pSVKeoX1, pSVZeoX1 and Zeo (Invitrogen) or pcDNA6/V5-His (Invitrogen), respectively. (B) Schematic pSVBsdX1 representation of the pIZKeoX1 vector. pIZKeoX1 is derived from pSVKeoX1 by the insertion of an IRES-lacZ-polyadenylation signal cassette from the Antibiotic Escherichia coli ES cells pBV.IRES.LacZ.PA plasmid (14). Detailed map and sequence information of the plasmids used are available on the Web site: http://www.embl-heidelberg.de/ µ pSVKeoX1 kanamycin 30 g/ml – ExternalInfo/stewart/plasmids.html G418 – 200 µg/ml pSVZeoX1 zeocin 25 µg/ml 25 µg/ml pSVBsdX1 blasticidin 50 µg/ml 5 µg/ml linearized DNA in an electroporation cuvette with a 0.4-cm 5.6 × 107 E14 ES cells were electroporated with 20 µgofScaI-linearized electrode gap. Cells were electroporated with a Bio-Rad Gene plasmids using a Bio-Rad Gene Pulser set at 240 V, 500 µF. The cells µ were plated onto four 10-cm gelatinized plates and, after 24 h, cells were Pulser set at 240 V, 500 F. The cells were plated onto four 10- fed with media additionally supplemented with different concentrations of cm gelatinized plates. After 24 h cells were fed with medium antibiotics. In parallel experiments, pSVKeoX1 and pSVBsdX1 gave a additionally supplemented with the suitable antibiotics (Table similar number of resistant colonies when compared to pMC1neopolyA 1). Southern analyses were performed by standard procedures. (1) as a standard, while pSVZeoX1 gave 10 times fewer colonies.

RESULTS ES cell culture and transformation As with previous ways to build targeting constructs, the E14 ES cells were grown in Dulbecco’s modified Eagle’s examples described here began with identification of mouse medium supplemented with 15% fetal calf serum, 100 µM non- genomic clones from isogenic λ DNA libraries (10) encompassing essential amino acids, 1 µM β-mercaptoethanol and leukemia the region to be targeted. Complete λDASH NotI-inserts were sub- inhibitory factor (LIF) (ESGRO™; Gibco BRL) at 37°Cina cloned into pZErO2.1 (Invitrogen). In contrast to previous λ humidity-saturated 9% CO2 atmosphere. The cells were cultured approaches, these complete subclones provided the back- on confluent feeder layers mitotically inactivated by treatment bone for the targeting construct. This presents the first merit of with mitomycin C except when they were grown in the presence our approach, namely the homology arms for recombination in of antibiotics. For transformation, E14 ES cells were ES cells were not subject to further manoeuvring, thus reducing trypsinized and resuspended in phosphate-buffered saline the possibility that they acquire inadvertant mutations. The (PBS) at a concentration of 1 × 107/ml. For each individual subclones were then modified by ET recombination using the transformation 0.8 ml cells (5.6 × 107) were mixed with 20 µg methodology described by Zhang et al. (4). ii Nucleic Acids Research, 1999, Vol. 27, No. 17 e16

Figure 2. Generation of a knock-out construct in a single step. (A) The general strategy. Linear DNA, synthesized by PCR to amplify zeocin resistance gene (ble) and including 50-nt homology arms (a and b) is co-transformed with a plasmid containing the target genomic DNA into a sbcA E.coli strain. Recombinants are identified by selection on zeocin. (B) Schematic representation of the Ssrp1 protein and gene. Position of the HMG box is shown. The isolated genomic clone contains the 17 exons of the Ssrp1 gene. The map of all the EcoRI restriction sites is indicated as well as the various EcoRI fragments (R1–R6) together with their size (in kb). The ble transcription unit synthesized by PCR from pSVZeoX1 is indicated at the bottom. ET recombination was performed in the sbcA JC8679 E.coli strain, and recombinants were isolated in the presence of 25 µg/ml zeocin (Invitrogen). (C) Agarose gel showing the EcoRI restriction pattern of the vector containing the genomic fragment of Ssrp1 (Genomic clone), and one of the recombinant products. The restriction fragments R1–R6 are shown as well as the EcoRI fragment corresponding to the pZErO2.1 vector. As expected, the restriction fragments R1 and R5 are missing, and a new 1.95 kb EcoRI fragment containing the ble transcription unit is present in the recombinant construct. (D) Southern blot analysis showing homologous recombination in E14 ES cells isolated in zeocin (25 µg/ml) after electroporation of the NotI-Ssrp1::zeo targeting DNA segment. Genomic DNA from wild-type (+/+) and targeted (+/–) E14 ES cells was cut with Asp718I and hybridized with a radiolabeled probe included in the R2 EcoRI restriction fragment. The wild-type allele is expected to give a 6.5 kb fragment and the targeted allele a 6.8 kb fragment (not shown).

In the first example, we explored the possibility that the E.coli and vertebrate cells. They are: (i) the neomycin resistance selectable gene required for ET recombination could then be gene (neo) which conveys kanamycin resistance in E.coli and used a second time as the selectable gene required for homo- G418 resistance in vertebrate cells; (ii) the bleomycin resistance logous recombination in ES cells, thereby greatly simplifying (ble) gene which conveys zeocin resistance in both E.coli and assembly of vertebrate targeting constructs. Currently there are vertebrate cells; and (iii) the blasticidin S deaminase gene three selectable genes that convey antibiotic resistances in both (bsd) which conveys blasticidin resistance in both E.coli and iii e16 Nucleic Acids Research, 1999, Vol. 27, No. 17

Figure 3. Combinational use of ET recombination and conventional in vitro DNA manipulations in a two-step process. (A) The general strategy. A first step of ET recombination is performed using PCR primers containing unique restriction sites for PmeIandAscI, flanking the chloramphenicol resitance gene (Cm). The resulting ET recombination product is purified and the restriction sites PmeIandAscI used to clone a suitable cassette to complete the targeting construct. (B) Schematic representation of the NSD2 protein and its different domains. The genomic clone used contains the Nsd2 gene from exon 10 to 18, encoding regions of the protein from the PHD fingers to the SET domain. The EcoRI restriction map is indicated as well as the various EcoRI fragments (R1–R5) together with their size (in kb). ET recombination products were obtained by chloramphenicol selection (100 µg/ml) in the sbcA JC8679 E.coli strain (recombinant 1). An IRES-lacZ- containing cassette was isolated from the pIZKeoX1 vector and then ligated into the ET recombination product using the PmeI/AscI restriction sites in order to generate the final targeting construct (recombinant 2). (C) Agarose gel showing the EcoRI restriction pattern of the construct containing the genomic fragment of Nsd2 (genomic clone), as well as the two types of recombinants (recombinants 1 and 2). The restriction fragments R1–R5 are shown as well as the EcoRI fragment corresponding to the pZErO2.1 vector. As expected, the EcoRI restriction fragments R4 and R5 are modified in the two recombined constructs. In the final targeting construct (recombinant 2), the R5 fragment is cut into a 900 bp fragment, R4 contains the neo transcription unit giving a 4.1 kb fragment, and a new 3.6 kb EcoRI fragment containing the IRES-lacZ sequence is obtained as predicted.

vertebrate cells. All three genes were cloned into a dual expression in E.coli and the SV40 early promoter (SVe) for expression in construct utilizing the E.coli bla promoter (blaP) for expression vertebrate cells (Fig. 1A). All three constructs conveyed the iv Nucleic Acids Research, 1999, Vol. 27, No. 17 e16 appropriate resistance in both E.coli, and in mouse E14 ES step a cassette containing an IRES-lacZ reporter and the cells (Table 1). selectable gene neo was isolated from the pIZKeoX1 plasmid To demonstrate the principle of the dual selection strategy, a (Fig. 1B), and cloned into the ET recombination product after subclone carrying 16.95 kb of the Ssrp1 gene was modified by cleavage of the introduced PmeIandAscI sites (Fig. 3). ET recombination (Fig. 2A). The genomic interval between Ssrp1 exons 6 and 16 was replaced with a PCR product made DISCUSSION from pSVZeoX1 using oligonucleotides that included ~60 nt of homology to either exon 6 or 16, by selection for zeocin ET recombination in E.coli was developed to engineer large resistance in E.coli. As described elsewhere (4,11,12), this ET DNA molecules, such as BACs, without restriction site recombination product was readily obtained (Fig. 2B and C). requirements (4,11,12). With two examples, we show the The ET product was directly used for targeting in ES cells by application of ET recombination to simplify previously difficult excising the 12.4 kb NotI DNA fragment containing the resistance engineering exercises. The examples concern the construction cassette flanked by 6 and 4.1 kb Ssrp1 homology arms. Out of of targeting vectors for homologous recombination in ES cells, eight zeocin-resistant ES clones analyzed, one exibited gene although the implications are also applicable to many engineering targeting at the Ssrp1 locus by homologous recombination exercises. The first example illustrates the generation of a (Fig. 2D). This experiment demonstrates that a knock-out targeting construct from a subcloned fragment of genomic construct can be made in a single step by ET recombination. DNA in a single, ET recombination, step. This example relies The dual resistance genes of the plasmids shown in Figure 1A are on the use of dual prokaryotic/eukaryotic promoters to express also flanked by loxP sites. Since we only wanted to knock-out antibiotic resistance from single coding regions. This is the Ssrp1, the PCR primers used to amplify the dual zeocin resistance simplest approach to construct a knock-out targeting vector yet gene of pSVZeoX1 excluded the loxP sites. However, for more described. The second example illustrates a two-step com- subtle applications (13) the loxP sites can be included by bination of new possibilities offered by ET recombination with choice of different PCR primers. the strengths of existing methodology. In the ET recombination In the first example, PCR was used to amplify a dual selectable step, unique restriction enzyme sites were introduced into the gene and the homology arms for ET recombination were subclone at freely chosen positions. In the second step, these encoded by the oligonucleotides used. In principle, it is possible to sites were used in a classical ligation subcloning to position a apply the same approach to amplify more complicated cassettes complex cassette between two genomic homology arms. In this that contain, in addition to a dual selectable gene, other elements approach, the final targeting construct did not contain PCR- such as a lacZ reporter gene. However, PCR is highly mutagenic. derived elements, hence this source of potential mutation was Whereas the functionality of the selectable gene is tested by the avoided. Other variations of two-step, or more, combinations of ET recombination step, the possibility that other elements ET recombination with classical restriction/ligation engineering, amplified by PCR are mutated is an undesirable risk that or PCR, are clearly possible. To date, we have successfully increases with size of the region to be amplified. To circum- applied similar strategies to generate targeting vectors for five vent this risk, we developed a second approach that combines genes without limitation. As described this approach allows the convenience of ET recombination with the most efficient targeting construct engineering within 1 week, which is faster conventional restriction/ligation methodology. In the second than using any other method, including those based on recA approach, ET recombination was used to position two restriction recombination in E.coli (14) or homologous recombination in enzyme sites exactly where desired. The two sites were yeast (15,16). Although the focus of this work has been the included in the oligonucleotides between the ET recombination fluent generation of targeting constructs for the mouse, the homology arms and the PCR primer for amplification of the principles described can be applied to any DNA engineering antibiotic resistance gene (Fig. 3A). Any gene that conveys exercise, whatever the purpose. antibiotic resistance in E.coli can be used in this application (not shown). Furthermore any restriction site(s) can be chosen ACKNOWLEDGEMENTS by this approach. Sites that will be unique in the product and convenient for the next subcloning step are selected. The product We wish to thank X. W. 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