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

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Simplified Generation of Targeting Constructs Using ET Recombination Pierre-Olivier Angrand, Nathalie Daigle1,Frankvanderhoeven,Hansr.Schöler1 and A © 1999 Oxford University Press Nucleic Acids Research, 1999, Vol. 27, No. 17 e16 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 restric- tion 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.
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