FEMS Yeast Research, 15, 2015, fov010 doi: 10.1093/femsyr/fov010 Advance Access Publication Date: 3 March 2015 Research Article

RESEARCH ARTICLE

A series of conditional shuttle vectors for targeted genomic integration in budding yeast Chia-Ching Chou, Michael T. Patel1 and Marc R. Gartenberg∗,2 Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University Piscataway, NJ 08854, USA ∗ Corresponding author: Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University Piscataway, NJ 08854, USA. Tel: +732-235-5800; E-mail: [email protected] 1 Present address: University of North Carolina, Eshelman School of Pharmacy, Chapel Hill, NC 27514, USA. 2 Member of The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA. One sentence summary: The authors describe a series of conditional shuttle vectors, each with CEN/ARS flanked by loxP sites, that can be used for both plasmid assembly in yeast and targeted genomic integrations. Editor: Pascale Daran-Lapujade

ABSTRACT The capacity of Saccharomyces cerevisiae to repair exposed DNA ends by homologous recombination has long been used by experimentalists to assemble plasmids from DNA fragments in vivo. While this approach works well for engineering extrachromosomal vectors, it is not well suited to the generation, recovery and reuse of integrative vectors. Here, we describe the creation of a series of conditional centromeric shuttle vectors, termed pXR vectors, that can be used for both plasmid assembly in vivo and targeted genomic integration. The defining feature of pXR vectors is that the DNA segment bearing the centromere and origin of replication, termed CEN/ARS, is flanked by a pair of loxP sites. Passaging the vectors through bacteria that express Cre recombinase reduces the loxP-CEN/ARS-loxP module to a single loxP site, thereby eliminating the ability to replicate autonomously in yeast. Each vector also contains a selectable marker gene, as well as a fragment of the HO locus, which permits targeted integration at a neutral genomic site. The pXR vectors provide a convenient and robust method to assemble DNAs for targeted genomic modifications. Keywords: CEN; ARS; Cre; loxP; shuttle vector; plasmid

INTRODUCTION An attractive feature of budding yeast Saccharomyces cerevisiae as a model organism is the remarkable proficiency that the genome can be modified by homologous recombination. Genes can be deleted, inserted and modified quickly and precisely. The ability to generate alterations solely with PCR products, thereby skipping traditional cloning in bacteria, has made yeast genomic modifications even more convenient. In these applications, a DNA segment with flanking regions of genomic homology ranging from 35–50 bps recombines with the chromosome via double crossovers (Oldenburg et al. 1997). The chromosomal DNA between the flanking homology regions is replaced by a gene conversion event.

Homologous recombination has also been used widely to assemble or alter plasmids in yeast (Ma et al. 1987). Typically, a shuttle vector is recombined with one or more DNA fragments to yield a plasmid that is subsequently recovered in bacteria for confirmation, amplification and storage. The interchangeable components of the pRS series of shuttle vectors are ideally suited for this purpose (Sikorski and Hieter 1989). Each pRS plasmid differs from the others by the identity of the selectable marker gene and/or by their mode of propagation in yeast. The extrachromosomal members of the series carry an autonomous replicating sequence (ARS) and either a centromere (CEN) or a 2 μM stability element that maintain the plasmids at low or high copy, respectively. Nevertheless, copy number of both types

Received: 5 December 2014; Accepted: 26 February 2015  C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]

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MATERIALS AND METHODS

ADE2 HIS3 TRP1 LEU2 URA3 MET15 LYS2 kanMX hphMX natMX pXRA2 pXRH3 pXRT1 pXRL2 pXRU3 pXRM15 pXRLy2 pXRkX pXRhX pXRnX

Length Marker Name

Table 1. pXR shuttle vectors.

The first plasmid of the pXR series, pXRL2 (Table 1), was assembled in the following steps. A loxP-CEN6/ARSH4-loxP fragment of plasmid pDS163 (Sinclair and Guarente 1997) was amplified by PCR (primers 1 and 2; Table 2) and joined to AatIIlinearized plasmid pRS405 by homologous recombination in yeast. The resulting plasmid pMTP1 was opened by partial digestion with HindIII and SalI, filled-in and reclosed to generate pMTP2. This plasmid was linearized with enzyme BsmBI and joined to three overlapping PCR products by homologous recombination in yeast to yield pXRL2. Two of the PCR products (amplified from strain W303-1A genomic DNA with primer combinations 5/6 and 7/8) recombined with one another to regenerate a 650 bp segment of the HO promoter with an AscI digestion site added to the center of the element (Fig. 1). The third PCR product (amplified from pMTP1 with primers 3 and 4) contained a replacement loxP site that lacked several redundant restriction sites. The remaining plasmids of the pXR series were generated by swapping the LEU2 marker of pXRL2 with ADE2, HIS3, TRP1, URA3, MET15 and LYS2 from other pRS vectors using PCR products generated with primers 9 and 10 (Sikorski and Hieter 1989; Brachmann et al. 1998; Eriksson et al. 2004). Additional pXR plasmids were generated by swapping LEU2 of pXRL2 with PCR products bearing drug-resistance marker genes kanMX from pNJ418K and hphMX from pNJ418H (gifts from Steve Brill) using primers 10 and 11. A final pXR plasmid was generated by swapping the kanMX marker of pXRkX with a natMX PCR product generated from plasmid pAG25 using primers 12 and 13 (Goldstein and McCusker 1999). Plasmid constructs were confirmed by

Partial list of unique RE sites in MCS

Plasmid and strain construction

KpnI, ApaI, XhoI, SalI, ClaI, EcoRI, PstI, SmaI/XmaI, BamHI, SpeI, EagI/NotI, SacII, SacI ApaI, XhoI, SalI, ClaI, EcoRI, SmaI/XmaI, BamHI, SpeI, EagI/NotI, SacII, SacI KpnI, ApaI, XhoI, SalI, ClaI, EcoRI, PstI, SmaI/XmaI, BamHI, SpeI, EagI/NotI, SacII, SacI ApaI, XhoI, SalI, PstI, SmaI/XmaI, BamHI, SpeI, EagI/NotI, SacII, SacI KpnI, XhoI, SalI, ClaI, EcoRI, SmaI/XmaI, BamHI, SpeI, EagI/NotI, SacII, SacI KpnI, ApaI, XhoI, SalI, ClaI, PstI, SmaI/XmaI, BamHI, SpeI, EagI/NotI, SacII, SacI ApaI, SalI, ClaI, EcoRI, PstI, SmaI/XmaI, EagI/NotI, SacI KpnI, ApaI, XhoI, SalI, EcoRI, SmaI/XmaI, BamHI, SpeI, EagI/NotI, SacII, SacI KpnI, ApaI, XhoI, SalI, ClaI, SmaI/XmaI, BamHI, SpeI, NotI, SacI ApaI, XhoI, SalI, ClaI, EcoRI, BamHI, SpeI, EagI/NotI, SacII, SacI

Partial list of unique RE sites in the marker gene

of plasmids can vary considerably between populations of cells and between single cells of a given population. Furthermore, spontaneous plasmid loss events yield a fraction of plasmid-free cells in every population, even under selective growth conditions (Murray and Szostak 1983). The integrative members of the pRS series lack both ARSs and stability elements. Instead they attain heritability by recombining into a chromosome. The fixed copy number of an integrative vector in every cell of a population is an attractive if not essential feature for a variety of genetic analyses and single cell studies. However, the greater challenge of assembling integrative vectors by homologous recombination in vivo represents a bottleneck in their broader application. While it is possible to join DNA fragments together efficiently during the integration process, the resulting DNA products cannot be recovered, further modified and reused because they have integrated (for example, see Kuijpers et al. 2013). We sought a method to harness the power of homologous recombination to assemble extrachromosomal plasmids that could ultimately be used for integration. To this end, we made a series of conditional shuttle vectors, each with a CEN/ARS element flanked by loxP target sites for the Cre site-specific recombinase. The extrachromosomal forms of the vectors facilitate the assembly of constructs by homologous recombination in yeast. Following isolation from yeast, the vectors are converted to their integrating forms by passaging through a bacterial strain that expresses Cre recombinase. A fragment of the HO locus was included in each vector to permit targeted integration at a neutral genomic site. This report documents the creation and application of conditional CEN/ARS shuttle vectors known as pXR vectors.

AatII, HpaI, StuI, NdeI, BsrGI NdeI, MscI, BsmI, NheI, NsiI SnaBI, MfeI, BsgI, BspMI HpaI, BstEII, AgeI, XcmI, BspMI, BsrGI NdeI, BsgI, SbfI, BspMI, XcmI, NcoI, BstBI, BsmI, StuI, NsiI NcoI, BspMI, NsiI, SnaBI StuI, HpaI, NruI, BglII N/A N/A N/A

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6725 5653 5471 6705 5581 6091 9270 5827 6046 5589

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Table 2. Oligonucleotides. Name

Sequence (5 -to-3 )

Application

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

tgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttcagctgaagcttcgtacgc tataggttaatgtcatgataataatggtttcttaggcataggccactagtggatctg ccatcattaaaagatacgaggcgcgtgtaagttacaggcaagcgatgcgagaacccttaatataacttcg gtgccgcttactcgtgagaatatcaaccttatagctagtggatctgatatcacctaataacttcg gctataaggttgatattctcacgag ggcgcgccatccagtacaatgcgaaacgctaccgataatggcaccgtcttttg tcggtagcgtttcgcattgtactggatggcgcgccttcagaaatttcacagttgatgaatc ctcccggcatccgcttacagacaagctgtgaccgtctccgggagatttctttgccagtaagaactac tatcacgaggccctttcgtc acagttgcgcagcctgaatg tatgcggcatcagagcagattgtactgagagtgcaccatagacatggaggcccagaatac ccttgacagtcttgacgtgc cgcacttaacttcgcatctg gtgtagaattgcagattcccttt gacggtcacagcttgtctgtaa aaccctatctcggtctattcttttg gttcatgtgtacaatgttcattatctc cagacaagctgtgaccgtct attaggtgatatcagatccactagc

Amplification of loxP-CEN6-ARSH4-loxP Amplification of loxP-CEN6-ARSH4-loxP New loxP New loxP Amplification of 5 -HOp-AscI Amplification of 5 -HOp-AscI Amplification of 3 -HOp-AscI Amplification of 3 -HOp-AscI pRS marker swap pRS marker swap MX marker swap Amplification of AgTEF2 promoter Amplification of AgTEF2 terminator Confirm genomic integration at LEU2 Confirm integration of marker gene Identify multiple integrations at marker gene Confirm genomic integration at HO Confirm genomic integration at HO Identify multiple integrations at HO

restriction digestion and sequencing of new junctions. The pXR vectors and their annotated sequence files are available from Addgene. Plasmid YCp-NLS-mCherry(LEU2) was generated in yeast strain GCY16 (W303–1A sir2::kanMX) by cotransformation with AlwNI and DraIII digested pXRL2 and ScaI and NdeI digested pBT054 (Timney et al. 2006). The sir2 strain was used on the presumption that the pseudo-diploid state, which promotes homologous recombination over end joining, would favor plasmid as˚ ¨ sembly (Astr om, Okamura and Rine 1999). In vivo assembly of clones, however, should work well in most common, wild-type, laboratory strains. YIp-NLS-mCherry was targeted for integration at HOp in yeast strain W303–1A by digestion with AscI. The same plasmid was targeted to leu2–3,112 in strain MRG5572 by digestion with ClaI to generate strain MRG5604. In each case, nine transformants were evaluated by PCR and found to contain YIP-NLS-mCherry at the intended target locus. Strain MRG5572 [MATa tS(CGA)C-BUD31::256lacop -TRP1 ADE2::HIS3pGFPlacI::ade2-1 leu2-3,112 lys2 (pWJ1327, CEN/URA3/NOP1-CFP)] was derived from strains described previously (Chen and Gartenberg 2014). The genotype of strain W303-1A is MATa ade21 can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3-1.

General procedures for using pXR plasmids Yeast were transformed with DNA by the LiOAc/DMSO method and individual transformants were isolated on selective agar plates (Soni, Carmichael and Murray 1993). Yeast plasmids were harvested from overnight cultures by glass bead lysis (Hoffman and Winston 1987) and introduced into either BNN132 or DH5α by electroporation using less than 1 μg of yeast genomic DNA extract/1 × 109 cfu of electrocompetent BNN132. We have found that lowering the DNA/BNN132 ratio reduces recovery of unrecombined material. When SwaI digestion was employed, DNA was desalted by ethanol precipitation before electroporation.

BNN132 and DH5α transformants were grown at 37◦ C in LB media with 50 μg/ml ampicillin to select for plasmids. Plasmid integrations in yeast were confirmed by fluorescence microscopy and/or by PCR. For integration at leu2, primer 14, which binds adjacent to the chromosomal LEU2 locus, was combined with primer 15 that binds to a site downstream of the marker gene in all pXR vectors. PCR with primers 15 and 16 that bind pXR plasmids on both sides of the marker genes was used to identify tandem integration events. For integration at HOp, primer 17, which binds the genome upstream of the HOp homology segment, was combined with primer 18 that binds all pXR vectors downstream of the HOp homology segment. PCR with primers 18 and 19 that bind the plasmid on both sides of the HOp homology segment was used to identify tandem integration events.

Microscopy Two milliliter cultures were grown overnight in selective SC-leu media with aeration and then back-diluted to 0.05–0.1 OD in 3 ml of fresh SC-leu for outgrowth to mid-log (0.4–0.6 OD). Cells were concentrated by microcentrifugation for two minutes at 6000 rpm and resuspended in 10–20 μl of water. Two microliters of cell suspension was pipetted onto 1.3% agarose plugs on microscope slides with depression wells (Fischer 50-949-458) and then sealed with a cover slip and nail polish (Rines et al. 2011). The agarose plugs contained SC media composed of synthetic nitrogen base that has negligible autofluorescence (Sheff and Thorn 2004). Z-stacks consisting of sequential images separated by 275 nm were collected with a Zeiss Axioplan II Fluorescence Microscope (100 × 1.4 NA objective). For costaining with 4 ,6-diamidino-2-phenylindole (DAPI), cells were fixed with formaldehyde (Chang et al. 2005) and then washed sequentially with 70% EtOH and H2 O before resuspending in PBS buffer containing 50 ng/ml DAPI. The images provided were composed from maximum intensity projections of several sequential layers.

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Figure 1. pXR vectors. Details of each vector are listed in Table 1. PCR primers for confirmation of integration (small numbered arrows) are listed in Table 2 and described in the section ‘Materials and Methods’.

Flow cytometry Cultures were grown to mid-log in selective media supplemented with 40 mg/L adenine sulfate. Samples were fixed with 4% paraformaldehyde (Chang et al. 2005) before resuspending in PBS. Flow cytometry was performed at the Rutgers EOHSI core facility on a Beckman Coulter Gallios flow cytometer (488 nm excitation laser, 620/30 nm emission filter) and analyzed with Kaluza software. Data were smoothed with a 5-point moving average using Microsoft Excel.

RESULTS AND DISCUSSION The pXR series of shuttle vectors The centerpiece of each pXR vector is a CEN6/ARSH4 element flanked by 34 bp loxP target sites for Cre recombinase from bacteriophage P1 (Sinclair and Guarente 1997). The loxP sites are oriented tandemly such that reaction with Cre results in excision of

the intervening DNA. Like other CEN/ARS plasmids, members of the pXR series propagate stably in yeast as low-copy extrachromosomal elements prior to Cre-mediated recombination. They serve as convenient backbones for plasmid manipulation by in vivo recombination, as described below. Following recombination to remove the loxP-CEN6/ARSH4-loxP element, the plasmids no longer replicate autonomously. However, regions of chromosomal homology within the plasmid permit targeted genomic integration. Each plasmid of the pXR series contains either a nutritional marker from S. cerevisiae or a dominant drug selection marker from bacteria (Fig. 1). Dominant drug selection markers offer the advantage that they do not change the biosynthetic profile of the recipient yeast strain (Pronk 2002). Nutritional markers, on the other hand, provide sequence homology for integration in appropriate auxotrophic strains. Some popular strains lack corresponding regions of homology to these genes. For example, the BY474 lineage bears deletions encompassing all of LEU2 and

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URA3 among other useful marker genes (Brachmann et al. 1998). To permit integrations that do not rely on sequence homology of a nutritional marker, a segment of the HO promoter was included in each pXR vector. In most laboratory strain lineages, HO is non-functional and therefore provides a generic, neutral site for integrating foreign DNA and sequence tags (see example in Yan et al. 2008). Linearization of the pXR vectors with AscI, which recognizes an 8 bp site engineered within the HO promoter fragment, targets integration of the vectors upstream of the HO gene on chromosome IV. The pXR plasmids were derived from the earlier pRS series of shuttle vectors. Thus, they also contain a bacterial origin of replication, a β-lactamase gene for selective growth in bacteria and other features of the pBLUESCRIPT precursor (Sikorski and Hieter 1989). A partial list of the unique restriction sites within the multiple cloning site (MCS) of each vector is provided in Table 1.

Cre recombinase-mediated conversion of pXR vectors To achieve Cre-mediated recombination, we passage pXR vectors through E. coli strain BNN132, which expresses Cre constitutively from a lysogenized λ phage (ATTC #47059; Elledge et al. 1991). After assembling a new pXR derivative in yeast, we prepare DNA extracts and introduce the extracts directly into BNN132 by electroporation. In our hands, 20% or more of the BNN132 transformants yield pure recombined plasmids that lack the CEN/ARS element. Typically, we screen 2–5 colonies. The remaining transformants contain mixtures of Cre-recombined and unrecombined plasmids, as shown below. The basis for this mosaicism is not clear but it has been reported by others (Stewart and Behringer 2010). To increase recovery of pure recombined plasmids, we digest yeast DNA extracts with SwaI, a restriction endonuclease that cuts within the CEN/ARS segment (Fig. 1). Cre-mediated recombination of the linearized vectors causes recircularization of the desired DNA and elimination of the CEN/ARS. After SwaI treatment, nearly 100% of BNN132 transformants contain pure recombined plasmids (see example below).

Application of the pXR shuttle vectors To demonstrate the utility of pXR vectors, we describe the assembly of an integrating vector that expresses mCherry fused to the nuclear localization signal of yeast Nab2 (Timney et al. 2006). In fluorescence microscopy studies of live cells, the NLSmCherry chimera provides an attractive alternative to DNA staining dyes like DAPI, which may sensitize DNA to UV damage and work best only after cell fixation (Hayashi et al. 1998). The NLS-mCherry expression cassette was released from its pRSbased parent vector with restriction endonucleases that preserve at least 30 bp of overlapping homology with a linearized pXR vector (pXRL2 with the LEU2 marker). In Fig. 2, the homology domains are labeled a and b (see the section ‘Materials and Methods’ for details). Transformation of yeast with both digests yielded more colonies on SC-leu plates than transformation with either digest alone. DNA isolated from the yeast transformants was passaged through either the Cre-expressing BNN132 strain or the traditional bacterial cloning strain DH5α. Restriction digestion patterns of plasmids recovered from DH5α confirmed that the NLS-mCherry cassette was transferred to pXRL2 (Fig. 3A, lane 2). Importantly, the digestion patterns of plasmids recovered from BNN132 showed that the loxP-CEN6/ARSH4-loxP element was removed from the construct in three out of five

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transformants (lanes 3–5). The remaining transformants contained mixtures of recombined and unrecombined plasmids (lanes with mixtures are highlighted with a circled lane number). Three additional independent transformations of BNN132 yielded similar results with 40–100% of the transformants bearing pure recombined product (Fig. 3B). Importantly, digesting the yeast extracts with SwaI before BNN132 transformation increased the recovery of pure recombined plasmids to 100% in each trial. The new CEN/ARS and integrating plasmids were named YCp-NLS-mCherry (LEU2) and YIp-NLS-mCherry (LEU2), respectively. YIp-NLS-mCherry (LEU2) was integrated at the HO promoter of strain W303–1A. Microscopy of fixed cells detected a fluorescent mCherry signal that broadly overlapped nuclear staining by DAPI (Fig. 4A). In the figure, spots of extra-nuclear DAPI fluorescence correspond to mitochondria where the DNA dye localized but NLS-mCherry did not (Shadel and Seidel-Rogol 2007). Conversely, nuclear regions with NLS-mCherry but not DAPI fluorescence correspond to nucleoli, where the DNA dye has been shown to fluoresce poorly (Shaw and McKeown 2011). To validate that NLS-mCherry reaches nucleoli, YIP-NLS-mCherry was integrated at the leu2 locus of a strain that expresses the nucleolar marker Nop1-CFP. Fig. 4B shows that the NLS-mCherry signal spans regions of the nucleus that also contain Nop1-CFP. Fluorescence microscopy of cells bearing YCp-NLS-mCherry (LEU2), the extrachromosomal CEN/ARS variant, yielded strikingly different results despite growth under selection for the plasmid. Nuclear mCherry fluorescence intensity varied from one cell to the next with some cells completely devoid of the signal (Fig. 4C). Similar results were obtained with a centromeric pXR derivative bearing the URA3 selectable marker (data not shown). Flow cytometry of the cultures confirmed that cells with YCp-NLS-mCherry (LEU2) yielded a broad distribution of fluorescence intensities (red trace; Fig 4D). Some cells with the CEN/ARS plasmid were brighter than cells with the integrated plasmid whereas others displayed background fluorescence no greater than cells that contained no plasmid. This cell-to-cell variation is one of the central disadvantages of using plasmid-borne expression vectors over their integrated counterparts.

Considerations and potential caveats We envision that the pXR vectors will benefit the yeast community by providing the convenience of in vivo recombination when assembling vectors for targeted genomic integrations. Typical applications might include the assembly of vectors for the stable expression of foreign genes, vectors for expression of endogenous genes at elevated copy number or traditional pop-in/pop-out vectors for scar-less genome modifications. The vectors should also be useful for assembling, confirming and then integrating chimeric genes and mutant alleles. Additionally and as shown here in Fig. 3, the pXR vectors provide a simple, ligase-free means to create integrative derivatives of centromeric plasmids that already exist in the laboratory. One limitation worth noting is that ARS elements sometimes reside within cloned yeast DNA (there are over 400 in the S. cerevisiae genome), as well as in DNA from other organisms (Siow et al. 2012; Liachko and Dunham 2014). Addition of these DNAs to pXR vectors will support autonomous plasmid replication even in the absence of a formal CEN/ARS element. We have maintained recombined plasmids in BNN132 as frozen glycerol stocks for several months. We know of no reason why longer-term storage in this strain would be problem-

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Figure 2. Flow chart for using pXR vectors. (Step 1) Transformation of yeast with a linearized pXR vector and one or more DNA fragments that contain domains of overlapping homology of at least 30 bp (labeled as a and b). Homologous recombination within yeast joins the fragments to form a hybrid plasmid. (Step 2) Preparation of yeast DNA extracts by glass bead lysis and recovery of the assembled plasmids in a bacterial strain that expresses Cre constitutively by electroporation. Cre reduces the loxP-CEN6/ARSH4-loxP module to a single loxP site. Optional step: digestion of yeast extracts with SwaI before transformation of Cre-expressing bacteria increases the recovery of pure recombined plasmids. (Step 3) Linearization of the resulting plasmid within regions bearing genomic homology promotes integration by homologous recombination. Targeting depends on where the double strand break is created. In this figure, breaks were created in either the HOp segment or the marker gene.

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Figure 3. Cre recombination of pXR derivatives in bacterial strain BNN132. (A) YCp-NLS-mCherry (LEU2) was generated from pXRL2 by in vivo recombination in yeast. After passaging through bacteria, isolated DNA was cut with XbaI. rec = YIp-NLS-mCherry (LEU2); unrec = YCp-NLS-mCherry (LEU2). Lane 1—pXRL2 isolated from DH5α; Lane 2—YCp-NLS-mCherry (LEU2) isolated from DH5α; Lane 3 through 7—Plasmid DNA isolated from BNN132 after transformation with yeast extracts bearing YCp-NLS-mCherry. Circled lane numbers correspond to isolates bearing mixtures of both YIp-NLS-mCherry (LEU2) and YCp-NLS-mCherry (LEU2). The 8 and 3 kb bands of an NEB 1 kb ladder are marked with single and double asterisks, respectively. (B) Three additional trials of BNN132 transformation with yeast extract bearing YCp-NLS-mCherry. Pre-digestion of extract with SwaI was compared to no pre-digestion.

atic. In practice, it is often desirable to obtain both the integrative version of a pXR vector, as well as the unrecombined pXR progenitor. Therefore, we typically recover a newly assembled vector from yeast with a traditional bacterial strain that lacks Cre, like DH5α, in addition to BNN132. Since individual yeast transformants can contain more than one plasmid species (Scanlon, Gray and Griswold 2009), care must be exercised when using parallel bacterial transformations to recover such related pXR derivatives. In our experience, off target events do not typically occur when using AscI linearization to direct integration of pXR vectors at HOp. In the event that a cloned insert contains an additional 8 bp AscI recognition sequence, partial digestion with the enzyme would be required. Note that the HOp element of pXR vectors is situated within a common pRS backbone. Prior genomic modifications with pRS vectors or other pBLUESCRIPT vectors thus provide decoy target sites for integration. Confir-

mation of clones by PCR with appropriate primers distinguishes integration at intended sites from integration elsewhere (see the section ‘Materials and Methods’).

ACKNOWLEDGEMENTS We thank Leonard Guarente, Mike Rout, David Stillman and Steve Brill for reagents and Derek Chen for technical assistance.

FUNDING This work was funded by the National Institutes of Health (GM51402 to MRG) and a summer fellowship from the New Jersey Commission on Cancer Research to MTP. Conflict of interest. None declared.

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Figure 4. Comparison of integrated and extrachromosomal pXR vectors bearing the NLS-mCherry expression cassette. (A) Strain W303–1A with integrated YIp-NLSmCherry (LEU2) after fixation and staining with DAPI. (B) Live cell images of strain MRG5604 with integrated YIp-NLS-mCherry and a plasmid expressing Nop1-CFP. (C) Strain W303–1A bearing YCp-NLS-mCherry (LEU2) after fixation and staining with DAPI. (D) Flow cytometry of strain W303–1A bearing YCp-NLS-mCherry (LEU2) or YIp-NLS-mCherry (LEU2) integrated at HOp after growth in SC-leu. W303–1A without an NLS-mCherry vector was included as a negative control after growth in SC media.

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A series of conditional shuttle vectors for targeted genomic integration in budding yeast.

The capacity of Saccharomyces cerevisiae to repair exposed DNA ends by homologous recombination has long been used by experimentalists to assemble pla...
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