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...30]) to Tubb3 was designed and products were amplified by PCR [31]. We also included a direct comparison with a standard yeast URA3 vector that contains extensive homology to the yeast genome, pRS316 =-=[32]-=-. In striking contrast to the zero background of pRTVIR upon single-tube transformation, pRS316 gave extensive background of yeast transformants and only modest increase in the number of transformants...

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...rming E. coli with the lyticase solution and selecting for ampicillin resistance. Following lysis, silica column binding, washing and elution, DNA was electroporated into E. coli cloning strain DH10B =-=[39]-=-. PAGP1-LacZ Recombination Experiments For experiments involving PCR products carrying flanking homology regions a 3.8 kb PAGP1-lacZ product was PCR amplified with Phusion DNA polymerase from YCpAGP1-...

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...ination (pRTVIR). (A) The sequence of Candida albicans Ura3 (CaUra3) modeled onto the crystal structure of Saccharomyces cerevisae orotidine-5-phosphate decarboxylase [28] using the the Phyre2 server =-=[44]-=-. The very C-terminal 6 residues of CaUra3 are part of a structurally important a-helix (marked in red). CaURA3 harboring yeast vector pCA771 (WT) was modified by sitedirected mutagenesis to remove, 2...

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...tech) and selection was performed on 20 mg/mL kanamycin plates. Primer sequences are included in Table S1. Differentiation TauEGFPhyg Cells Differentiation of mouse ES was carried out as described in =-=[42]-=-. Briefly, 56105 cells were plated on 6 well dishes in N2B27 neural differentiation medium. Media was changed every day for 8 days and cells were fixed and stained with anti-GFP (Aves Labs) and anti-T...

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...es (pRTVIR) Facilitates the Construction of Targeting Vectors for Embryonic Stem Cells Cloning overlapping DNA fragments in yeast has been established for rapid cloning of mammalian targeting vectors =-=[23]-=-. This methodology relies on the homologous recombination between mammalian DNA sequences and a shuttle plasmid vector that can replicate and be selected for in both yeast and Escherichia coli. We set...

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...ns in mouse ES cells have already been made [2,3]. Indeed, the first encyclopedia of mutations in the mouse is near to completion, largely due to productivity gains in serial recombination techniques =-=[4]-=-. Although knockout and conditional ablation of genes are the first steps in understanding gene function in vivo, allelic variants such as disease-mimicking point mutations or whole gene replacement (...

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...s recombination in yeast (Ura+) between a 3.4 kb PCR product (primers A and B (Table S1)) encompassing 2 micrometer sequences, ampicillin marker and E. coli origin of replication from template pRS423 =-=[35]-=- and a 1.2 kb PCR product encompassing PTEF-CaURA3 from template pUG60 [36] (primers C and D). CaURA3 deletion variants of pCA771, including pRTVIR, was constructed by site-directed mutagenesis using ...

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...and is a spontaneous Ade- derivative of [Cir0] strain yAF7 [37] with the genotype (MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1). Yeast was transformed by an adapted protocol described by =-=[38]-=-. Briefly, 10 ml CAY1179 was grown over night in 26YPD medium at 30uC. In the morning the strain was diluted in fresh 26YPD medium (3 ml per transformation) and grown at 30uC from starting OD600= 0.1....

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...mg pRS316 linearized with SacI/KpnI (New England Biolabs) or pRTVIR (HindIII/EcoRV; New England Biolabs) and transformed into yeast as described above. T2A sequences to generate separate polypeptides =-=[41]-=- were introduced at ends with heterologous sequences to avoid incorrect recombination. Special care should be taken to avoid DNA crosslinking by UV-light, a blue-light gel analysis table was used for ...

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...ized at unique HindIII and EcoRV restriction sites introduced right at the 39 end of the CaURA3DKKTGQL coding sequence and was together with a 3.8 kb PCR product encoding a LacZ reporter (PAGP1-LacZ) =-=[29]-=- used to cotransform [Cir0] ura3 yeast to Ura+. The PCR primers for PAGP1-LacZ had been designed so that they introduced the required KKTG codons together with 30 bp and 35 bp of Figure 1. Constructio...

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...: The authors have declared that no competing interests exist. * E-mail: joel.schick@helmholtz-muenchen.de . These authors contributed equally to this work. Introduction Since its first demonstration =-=[1]-=-, gene targeting in embryonic stem (ES) cells has evolved to be a routine technique to modulate gene function in vivo. The sequential steps of genomic DNA isolation, vector construction, homologous re...

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...t, we designed a vector for recombination cloning without any sequence homology to the yeast genome. Briefly, an heterologous auxotrophic selection marker CaURA3 (orotidine-5-phosphate decarboxylase) =-=[27]-=- from Candida albicans driven by the TEF promoter from Ashbya gossypii was combined with replication sequences derived from the yeast episomal 2 micrometer plasmid. E. coli replication sequences and a...

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...: Andréasson C, Schick AJ, Pfeiffer SM, Sarov M, Stewart F, et al. (2013) Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells. PLoS ONE 8=-=(9)-=-: e74207. doi:10.1371/journal.pone.0074207 Editor: Yann Herault, IGBMC/ICS, France Received May 2, 2013; Accepted July 30, 2013; Published September 13, 2013 Copyright: 2013 Andréasson et al. This ...

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... B (Table S1)) encompassing 2 micrometer sequences, ampicillin marker and E. coli origin of replication from template pRS423 [35] and a 1.2 kb PCR product encompassing PTEF-CaURA3 from template pUG60 =-=[36]-=- (primers C and D). CaURA3 deletion variants of pCA771, including pRTVIR, was constructed by site-directed mutagenesis using oligonucleotides (E, D and F). pRTVIR and complete sequences of this plasmi...

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... DNA fragments, a five-fragment mammalian targeting vector for targeting a Tau-EGFP and hygromycin fusion (with adjoining T2A sequences; [30]) to Tubb3 was designed and products were amplified by PCR =-=[31]-=-. We also included a direct comparison with a standard yeast URA3 vector that contains extensive homology to the yeast genome, pRS316 [32]. In striking contrast to the zero background of pRTVIR upon s...

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...s are often not optimal for the construction of isogenic targeting constructs. For example, bacteriophage lambda-red based homologous recombination in bacteria has simplified genomic DNA modification =-=[19]-=-, but has the prerequisite of a library of mapped BACs or plasmid vectors as a source of genomic DNA. The library requirement hinders its application in, for instance, targeting campaigns of unique hu...

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...ery day for 8 days and cells were fixed and stained with anti-GFP (Aves Labs) and anti-Tubb3 (Abcam) to confirm co-localization. ES Cell Electroporation Electroporation of E14TG2a (ATCC) and JM8A1.N3 =-=[43]-=- cells was carried out as described [4]. The exceptionally high gene targeting frequency is due to a well-established targeting pipeline in projects contributing to the International Knockout Mouse Co...

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...R functions in homologous recombination of multiple overlapping DNA fragments, a five-fragment mammalian targeting vector for targeting a Tau-EGFP and hygromycin fusion (with adjoining T2A sequences; =-=[30]-=-) to Tubb3 was designed and products were amplified by PCR [31]. We also included a direct comparison with a standard yeast URA3 vector that contains extensive homology to the yeast genome, pRS316 [32...

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... chimaera production have seen increases in efficiency that have accelerated the number of new mutants each year such that more than 16,000 targeted mutations in mouse ES cells have already been made =-=[2,3]-=-. Indeed, the first encyclopedia of mutations in the mouse is near to completion, largely due to productivity gains in serial recombination techniques [4]. Although knockout and conditional ablation o...

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...ency or changes in expression level that may preclude correct targeting. It has been reported that even a 2% strain difference between targeting constructs can lead to a .25 fold in targeting success =-=[34]-=-, yet polymorphisms are not expected to act in favor of the non-isogenic construct. Targeting the 39 UTRs of genes may also lead to a bias, as they are known to be more highly polymorphic than coding ...

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...nstructs Tubb3-TauEGFP targeting construct. PCR amplification was used to generate fragments corresponding to the 9 kb flanking genomic DNA in 59 and 39 to the stop codon as well as TauEGFP (pTauEGFP =-=[40]-=-), hygromycin (pSectag) and PGK-neomycin (EUCOMM resource), gel-purified and combined with 1 mg pRS316 linearized with SacI/KpnI (New England Biolabs) or pRTVIR (HindIII/EcoRV; New England Biolabs) an...

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...ny formation frequency and a six-codon deletion (DKKTGQL) completely abolished transformation to Ura+ (Fig. 1A). Inspection of a structure model of CaUra3 based on the structure of S. cerevisiae Ura3 =-=[28]-=- revealed that the deletions had truncated an alpha-helix that is important for the structural integrity of the orotidine-5-phosphate decarboxylase enzyme. We named the new vector pRTVIR, plasmid for ...

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... chimaera production have seen increases in efficiency that have accelerated the number of new mutants each year such that more than 16,000 targeted mutations in mouse ES cells have already been made =-=[2,3]-=-. Indeed, the first encyclopedia of mutations in the mouse is near to completion, largely due to productivity gains in serial recombination techniques [4]. Although knockout and conditional ablation o...

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...species relevance or for clinical applications, these mutations are increasingly created in human ES or iPS cells [5–10], a process that is still somewhat tedious due to its use in niche applications =-=[11]-=-. Nonetheless, paired with new technologies to increase homologous recombination such as zincfinger nucleases, TALens, and CRISPR [12–15], targeting vectors are employed widely for genome modification...

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... DNA, thus effectively eliminating background arising from homologous recombination. Second, we wanted to eliminate background arising from in vivo ligation, which in yeast occurs with high frequency =-=[21]-=- by making the functionality of the CaURA3 selection marker in pCA771 conditionally dependent on a successful recombination event. Briefly, we made small 39 truncations of the CaURA3 gene of our pCA77...

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...ragment cloning in the yeast Saccharomyces cerevisiae, in which short homologous sequences are joined by the DNA double-strand break repair machinery [21–24]. In particular, larger homologous regions =-=[25]-=- or overlapping oligomers [26] have been successfully used to retrieve genomic DNA from BACs for the introduction into targeting constructs carried on yeast shuttle vectors. Importantly, yeast readily...

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...accharomyces cerevisiae, in which short homologous sequences are joined by the DNA double-strand break repair machinery [21–24]. In particular, larger homologous regions [25] or overlapping oligomers =-=[26]-=- have been successfully used to retrieve genomic DNA from BACs for the introduction into targeting constructs carried on yeast shuttle vectors. Importantly, yeast readily recombines multiple fragments...

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...irement hinders its application in, for instance, targeting campaigns of unique human iPS cell lines or in patient-specific gene-therapy, although substantial improvements have been made here as well =-=[20]-=-. Moreover, the lambda-red methodology requires sequential steps in targeting vector construction. Alternative methods generally employ multiple-step PCR, which is challenging for longer sequences fro...

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...mid are available from Addgene (Addgene ID 46365). Yeast Transformation and Plasmid Rescue Yeast strain CAY1179 was used for all experiments and is a spontaneous Ade- derivative of [Cir0] strain yAF7 =-=[37]-=- with the genotype (MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1). Yeast was transformed by an adapted protocol described by [38]. Briefly, 10 ml CAY1179 was grown over night in 26YPD medi...