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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: Mol Biotechnol. 2013 Jun;54(2):504–514. doi: 10.1007/s12033-012-9591-y

A Low-Copy-Number Plasmid for Retrieval of Toxic Genes from BACs and Generation of Conditional Targeting Constructs

Giyoun Na 1, Andrew Wolfe 2, CheMyong Ko 3, Hyesook Youn 4, Young-Min Lee 5,6, Sung June Byun 7, Iksoo Jeon 8, Yongbum Koo 9,✉
PMCID: PMC4888602  NIHMSID: NIHMS785515  PMID: 22945876

Abstract

Bacterial Artificial Chromosome (BAC) clones are widely used for retrieving genomic DNA sequences for gene targeting. In this study, low-copy-number plasmids pBAC-FB, pBAC-FC, and pBAC-DE, which carry the F plasmid replicon, were generated from pBACe3.6. pBAC-FB was successfully used to retrieve a sequence of a BAC that was resistant to retrieval by a high-copy-number plasmid via λ Red-mediated recombineering (gap-repair cloning). This plasmid was also used to retrieve two other genes from BAC, indicating its general usability retrieving genes from BAC. The retrieved genes were manipulated in generating targeting vectors for gene knockouts by recombineering. The functionality of the targeting vector was further validated in a targeting experiment with C57BL/6 embryonic stem cells. The low-copy-number plasmid pBAC-FB is a plasmid of choice to retrieve toxic DNA sequences from BACs and to manipulate them to generate gene-targeting constructs by recombineering.

Keywords: Low-copy-number plasmid, Bacterial artificial chromosome, Toxic gene, Recombineering, Targeting vector

Introduction

Recombinogenic engineering or recombineering (recombination-mediated genetic engineering) technique was developed for DNA manipulation to circumvent several limitations, such as size and available restriction sites, that are often encountered in conventional gene manipulation which utilizes restriction enzymes and DNA ligase [1]. The use of Red recombinase of phage λ, which requires a short homology (>45 bp), has become a powerful tool in genetic engineering [2]. This technique has made it possible to clone and modify specific regions or sites of a DNA molecule by recombineering. Together with the use of this homologous recombination process, the use of site-specific recombination systems, such as Cre-loxP and Flp-FRT, has expanded the range of applications of recombineering [3, 4]. Mostly, this technique has been employed to construct targeting vectors for mouse genome modification, including gene knockout [3, 5, 6], point mutation [7, 8], and gene knock-in [9].

The first step in making a targeting construct is to isolate a genomic DNA clone that carries the gene of interest from a genomic library. This step can be skipped for mouse genes, because mouse bacterial artificial chromosome (BAC) clones whose gene maps can be browsed online (http://www.ensembl.org) are available from CHORI (http://bacpac.chori.org) and Geneservice (http://www.geneservice.co.uk). A gene of interest in a BAC clone is cloned into a plasmid and then modified by recombineering [10], or BAC DNA is modified in situ by recombineering, and then DNA containing the modified region is cloned into a plasmid vector [11].

Plasmids for targeting constructs are mostly high-copy-number vectors. However, they often become unstable or kill the hosts due to their high gene dosage effects when expressing toxic sequences. We tried to retrieve the mouse gene kiss1r, which codes for GPR54 or the kisspeptin receptor, in a high-copy-number plasmid, pL253 (derived from pBluescript) by homologous recombination (gaprepair cloning) [3], but this attempt was unsuccessful. In order to circumvent this difficulty, a low-copy-number plasmid vector pBAC-FB was developed (GenBank accession no.:HQ670402, 9.67 kb, chloramphenicol resistance (CamR) and ampicillin resistance (AmpR)) derived from pBACe3.6 (GenBank accession no.: U80929, 11.5 kb, CamR) [12], which is often used for BAC library construction. A DNA region containing kiss1r, which seemed to carry a cryptic sequence toxic to Escherichia coli, was successfully retrieved from BAC clones into this plasmid. Two more DNA regions, which encode FK506-binding protein 52 (coded by fkbp4 gene) and FK506-binding protein 51 (coded by fkbp5 gene), respectively, were also successfully retrieved into this plasmid. The advantages of using this low-copy-number plasmid in recombineering-based construction of targeting vectors are discussed.

Materials and Methods

Strains and Plasmids

E. coli SW106 carrying a defective λ prophage and arabinose-inducible Cre, pL253 (5.35 kb, AmpR), a loxP-Neo-loxP (LNL) cassette vector pL452 (4.82 kb, AmpR), and a FRT-Neo-FRT-loxP (FNFL) cassette vector pL451 (4.83 kb, AmpR) were provided by the National Cancer Institute at Frederick (NCI-Frederick) (Frederick, MD, USA). pBACe3.6 was used to make the low-copy-number vectors pBAC-FB and pBAC-FC (GenBank accession no.: HQ670403, 9.26 kb, AmpR). E. coli TOP10 was purchased from Invitrogen (Carlsbad, CA, USA) and was used for routine transformation and plasmid preparation. A BAC clone (clone ID; bMQ-325-K6) containing kiss1r was obtained from Geneservice (Cambridge, UK). The BAC clones RP23-23B10 and RP23-34F5, containing fkbp4 andfkbp5, respectively, were obtained from Invitrogen (Carlsbad, CA, USA).

Electroporation

E. coli SW106 was grown to log phase (OD600 = 0.5) in Luria-Bertani (LB) broth (1 % tryptone, 0.5 % yeast extract, 0.5 % sodium chloride, pH 7.0) supplemented with 50 µg/ml of ampicillin (Amp) or 12.5 µg/ml of chloramphenicol (Cam). In order to induce Red recombinase expression, the culture was incubated at 42 °C for 15 min. The heat-treated culture was cooled on ice for 10 min, and cells were harvested from 6 ml by centrifuging at 6,000×g. The harvested cells were washed three times with 1 ml of sterile pure water (18.3 MΩ) and resuspended in 20 µl of sterile pure water. Cells were mixed with 25 ng of DNA. Electroporation was carried out in a cuvette with a 0.15 cm gap at 2,400 volts using Cell-Porator with Voltage Booster (Gibco-BRL, Bethesda, MD, USA). In order to induce Cre expression, arabinose was added to a final concentration of 1 mM for 1 h.

Generation of the Low-Copy-Number Plasmids pBAC-FB, pBAC-FC, and pBAC-DE

pBACe3.6, which contained an F plasmid replicon, was used to construct pBACE-FB and pBAC-FC. The coding region of the AmpR gene of the pGEM3Zf(+) vector (Promega, Madison, WI, USA) was amplified by polymerase chain reaction (PCR) using the primer pair BLA-F and BLA-R (Table 1) and was used to replace the AscI–NotI fragment, which contains the attTn7, PI-SceI, and loxP511 regions of pBACe3.6, to give AmpR. The pUC-link, the SacBII-coding region, and a loxP sequence of pBACe3.6 were replaced with the NotI-BstEII fragment, which contains the negative selection marker (herpes simplex virus thymidine kinase (HSV-tk)), from the high-copy-number plasmid pL253 to generate pBAC-FB (CamR, AmpR). pBAC-FC (AmpR) was generated by deleting the majority of the chloramphenicol acetyl transferase coding region of pBAC-FB by BsmI digestion and religation. pBAC-DE (GeneBank accession no.: HQ670404, 8.82 kb, CamR) was generated by deleting pUC-link from pBACe3.6 by EcoRI digestion and re-ligation.

Table 1.

Synthetic primer pairs used for polymerase chain reaction

Primer pairsa Sequence Homology Use
GPR54A, GPR54B 5′-GCGGCCGCTGCTCACAGCCTGGTCTC-3′ Left Gene retrieval
5′-AAGCTTGGCTCAGACTTTGTCCTCAG-3′
GPR54Y, GPR54Z 5′-AAGCTTGTGATACACTTCGTGATTCATGA-3′ Right
5′-GGATCCGTCAGCAAGTGCCTTTACCTTC-3′
FKBP4-1F, FKBP4-1R 5′-GCGGCCGCCAATCAGCAAGTGCTGGGTTC-3′ Left
5′-AAGCTTCTAAGACACCTCCACCTCAAG-3′
FKBP4-2F, FKBP4-2R 5′-AAGCTTGTGAGAAAGTATAGTCTGGTGTG-3′ Right
5′-GGATCCACTGATATGAAAGAGAAGGATTG-3′
FKBP5-1F, FKBP5-1R 5′-GCGGCCGCTGTCCTGGAACTCACTCT-3′ Left
5′-AAGCTTCAGACATGTAATACAAGTGGAG-3′
FKBP5-2F, FKBP5-2R 5′-AAGCTTGTACAGCACCACACGTTTGTT-3′ Right
5′-GGATCCCTCAAGGGTGTTAAAGTTGTCTG-3′
FKBP4-3F, FKBP4-3R 5′-GTCGACCAATGCCTGACCGATAAGTTCT-3′ Left LNL insertion
5′-GAATTCGACTCGGCTGCACCCACT-3′
FKBP4-4F, FKBP4-4R 5′-GGATCCCATCCAGTATGTGTCTTTGGG-3′ Right
5′-GCGGCCGCCAACTTCGGAGCCTATCTATC-3′
FKBP4-5F, FKBP4-5R 5′-GTCGACCATCCAGTATGTGTCTTTGGG-3′ Left FNFL insertion
5′-GAATTCCAACTTCGGAGCCTATCTATC-3′
FKBP4-6F, FKBP4-6R 5′-GGATCCATAGCAGCTGTCACAAGAAAAAGT-3′ Right
5′-GCGGCCGCGAGTGCTGGGATTAAAGGCGT-3′
BLA-F, BLA-R 5′-GGCGCGCCAAGAAGATCCTTTGATCTT TTCTAC-3′ Ampicillin resistance
5′-GCGGCCGCGCACTTTTCGGGGAAATGTG-3′
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a

GPR54, FKBP4, FKBP5, and BLA stand for the relevant genes, kiss1r, fkbp4, fkbp5, and bla(beta-lactamase), respectively

Subcloning of BAC DNA by Recombineering (Gap-Repair Cloning)

The mouse kiss1r gene fragment that was retrieved from the BAC clone was 8.95 kb long and contained exons 1–5. The mouse fkbp4 and fkbp5 gene BAC DNA fragment included exons 1–5 and exons 3–4, respectively, and spanned 8.13 and 8.21 kb, respectively. All of the recombineering procedures were carried out as previously described [3]. The low-copy-number targeting plasmid pBAC-FB was used for the recombineering-based cloning of DNA regions from BAC clones. Two end homologies (left-end homology, NotI/HindIII fragment; right-end homology, HindIII/BamHI fragment) were amplified by PCR using primer pairs (Table 1) and BACs as templates. The amplified sequences were cloned into pBAC-FB between the BamHI and NotI sites. The plasmid was linearized by digesting for 2 h with a threefold excess of HindIII to produce a gapped plasmid. The gapped plasmid (25 ng) was electroporated into heat-induced E. coli SW106 cells (20 µl) carrying BAC. After 1 h of incubation in 1 ml of LB broth, the transformants were selected on LB plates supplemented with 50 µg/ml of Amp (LB+Amp plate) at 32 °C for 24 h. Single colonies were streaked (approximately 1 cm long for each colony) on an LB+Amp plate with a sterile toothpick and the plates was incubated at 32 °C for 24 h. Colony cracking analysis was performed to check for successful subcloning as described elsewhere [13]. Briefly, bacterial cells equivalent to 3 or 4 colonies were transferred to a microtube by a toothpick and were resuspended in 25 µl of 10 mM EDTA. Twenty-five microliters of cracking buffer (0.2 M sodium hydroxide, 0.5 % sodium lauryl sulfate, and 20 % sucrose) was added, and the mixture was vortexed briefly and incubated at 70 °C for 5 min. After cooling at room temperature for 5 min, 0.75 µl of 4 M potassium chloride was added, and the mixture was vortexed briefly and incubated on ice for 5 min. After centrifuging at 13,000 rpm at 4 °C for 3 min, the supernatants (10–20 µl) were electrophoresed along with the supernatant from cells with non-recombinant pBAC-FB as a marker on a 0.7 % agarose gel at 2.5 V/cm for 2 h. Colonies carrying recombinant plasmids were selected and used for plasmid isolation with a QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany). The inserts were confirmed by restriction digestion of the purified plasmids and finally by nucleotide sequencing of both ends with the sequencing primers FB-F (5′-CAGGGTTATTGTCTCATGAGC-3′) and FB-R (5′-CGCCAATGACAAGACGCTG-3′).

Construction of a Conditional Knockout Targeting Vector

A conditional knockout targeting vector for fkbp4 was constructed from pBAC-FB-fkbp4 by recombineering according to the method previously described [3]. Our strategy was to insert one loxP site upstream of exon 2 and another loxP downstream of exon 2 to generate a frame shift by Cre-mediated deletion of exon 2 in the target genome. The upstream loxP site was inserted as described below. Two homology sequences, corresponding to the left and right homology sequences of the first loxP insertion site, were amplified by PCR using the primer pairs FKBP4-3F/FKBP4-3R and FKBP4-4F/FKBP4-4R. The left homology sequence was cloned into pL452 between the SalI and EcoRI sites, and the right homology was cloned into pL452 between the BamHI and NotI sites. The LNL cassette with the homology sequences at both ends was cut out with SalI and NotI, and run on a 0.7 % agarose gel at 2.5 V/cm for 2 h. This DNA fragment was then gel-purified and electroporated into heat-induced E. coli SW106 carrying pBAC-FB-fkbp4. Cells containing the recombinant plasmid pBAC-FB-fkbp4-LNL (map not shown) were selected on an LB plate containing 25 µg/ml of kanamycin (Kan) (LB+Kan plate). The E. coli SW106 carrying pBAC-FB-fkbp4-LNL was grown in LB+Kan broth until the OD600 was 0.5. Then Cre expression was induced for 1 h by adding 1 mM arabinose to delete the sequence between the two loxPs of the LNL cassette leaving just one loxP. The cells were serially diluted (103–106 folds), and 0.1 ml of each dilution was spread on an LB plate containing ampicillin. Colonies were picked and transferred to an LB+Kan plate to check sensitivity to Kan. Clones containing the recombinant plasmid pBAC-FB-fkbp4-L (AmpR, kanamycin-sensitive) (map not shown) were selected and used for the insertion of the FNFL cassette. The two end homologies that were amplified by primer pairs, FKBP4-5F/FKBP4-5R and FKBP4-6F/FKBP4-6R, were cloned at both ends of the FNFL cassette of pL451. The FNFL cassette was cut with SalI and NotI and then gelpurified. In order to insert the FNFL cassette into intron 2, the gel-purified FNFL cassette was electroporated into heat-induced E. coli carrying pBAC-FB-fkbp4-L. Cells with the recombinant plasmid pBAC-FB-fkbp4-FNFL were selected on an LB+Kan plate. The presence of loxP was confirmed by nucleotide sequencing using the gene-specific sequencing primer (5′-CTAAGCTAATCACTGGTATC-3′). The position of the FNFL cassette in the final construct was confirmed by restriction analysis and nucleotide sequencing using the sequencing primers FN-F (5′-TGGGCTCTATGGCTTCTGA-3′) and FN-R (5′-ATGCTCCAGACTGCCTTG-3′).

Incompatibility Test

Incompatibility between a BAC (CamR, carrying mouse fkbp4) and retrieval plasmids was tested by electroporating a retrieval plasmid (pL253 or pBAC-FC) into E. coli SW106 cells carrying the BAC plasmid and then selecting transformed cells on LB+Amp plates. After 24 h of incubation at 32 °C, a single colony (1–2 mm in diameter) was selected with end-cut yellow tips, resuspended in 1 ml of LB broth, and serially diluted to 101–103 folds. Aliquots (100 µl) of the diluted cell suspension were spread on LB+Amp plates to count the total number of cells in a colony, and an LB plate containing Cam (12.5 µg/ml) (LB+Cam plate) was used to count cells carrying the BAC and the retrieval plasmid. Incompatibility between pL253 and pBAC-DE and between pBAC-DE and pBAC-FC was also tested.

Gene Targeting in Embryonic Stem Cells

C57BL/6 embryonic stem cells (American Type Culture Collection, Manassas, VA, USA) were cultured on a mitomycin-treated mouse embryonic fibroblast feeder layer. The embryonic stem cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15 % fetal bovine serum (Millipore, Billerica, MA, USA) and 1,000 units/ml of leukemia inhibitory factor (Millipore, Billerica, MA, USA). The targeting plasmid pBAC-FB-fkbp4-FNFL was purified using a QIA filter Plasmd Mega Kit (Qiagen, Valencia, CA, USA) and was digested with Not1 for electroporation. Electroporation of embryonic stem cells was carried out as described [14]. 5 × 106 cells were electroporated with 50 µg of linearized targeting vector. Electroporated cells were plated onto feeder layers. Drug selection (G418, 300 µg/ml; ganciclovir, 1 µM) was started after 1 day. After 12 days of selection, G418-resistant cell colonies were picked and expanded. DNA extraction was carried out as described [14]. Homologous recombination at the short arm homology region was confirmed by PCR. PCR reactions were carried out with primers, 5′-CTATACGAAGTTATTAGGTGGATC-3′ (specific to the incoming FNFL) and 5′-GGTCCTTGTGGTAGCCTTC-3′ (specific to the target locus). PCR was performed in a total volume of 50 µl containing 200 ng of genomic DNA, 200 µM of deoxyribonucleoside triphosphates, 0.2 µM of each primer, 2.5 units of HS prime Taq DNA polymerase (Genet Bio, Nonsan, Chungnam, Korea), 10 mM Tris–HCl (pH 8.3), 50 mM KCl, and 1.5 mM MgCl2. Amplification was performed with an initial denaturation for 10 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min, and extension at 72 °C for 1 min.

Results

Generation of Retrieval Plasmids

Two low-copy-number vectors for the use of retrieving target genes from BAC clones were generated. Both vectors carry the same multiple cloning sites and a negative selection marker, HSV-tk, which was derived from the high-copy-number plasmid pL253. The attTn7 [15], PI-SceI [16], a mutated loxP (loxP511) [17], and a loxP site that were present in pBACe3.6 were eliminated in these vectors. They are named as pBAC-FB that is CamR and AmpR (Fig. 1a) and pBAC-FC that is AmpR (Fig. 1b).

Fig. 1.

Fig. 1

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Circular maps of the retrieval plasmids pBAC-FB and pBAC-FC. Both plasmids carry F plasmid replicons of pBACe3.6. pBAC-FB contains chloramphenicol resistance and ampicillin resistance genes. pBAC-FC has only an ampicillin resistance gene. Both plasmids have single recognition sites for NotI and BamHI that can be used for cloning end homology sequences

Retrieving Target Regions from BAC Using the Low-Copy-Number Targeting Vector pBAC-FB

Retrieving the mouse genomic kiss1r (8.95 kb) gene from a BAC into the high-copy-number vector pL253 by recombineering was unsuccessful for an unknown reason. The toxicity of eukaryotic genomic sequences, especially in high-copy-number vector, was a suspected cause of plasmid instability in E. coli. Plasmid instability is known to occur due to the presence of a cryptic promoter that produces a toxic peptide from eukaryotic genomic sequences [18, 19]. Therefore, the low-copy-number vector pBAC-FB was selected instead of the high-copy-number plasmid pL253 to reduce the possible toxic effects of the genomic sequence containing kiss1r in E. coli. By electroporating linear (HindIII-cut) pBAC-FB with end homologies into the heat-induced E. coli SW106 carrying BAC, several hundred colonies appeared on an LB+Amp plate. Colony cracking analysis of 10 transformants showed that clones 1 (lane 1) and 9 (lane 9) were found to contain recombinant plasmids that retrieved the DNA region containing kiss1r (Fig. 2a). Some colonies displayed no visible plasmid bands in the colony cracking analysis. In order to test whether pBAC-FB can be generally used for gene retrieval from BACs, regardless of gene toxicity, gene retrieval was carried out for other non-toxic sequences containing fkbp4 (8.13 kb) and fkbp5 (8.21 kb), cloned into BACs. Colony cracking analysis indicated that gene retrieval was successful for these sequences (Fig. 2b, c). In colony cracking analysis, clones 2 (lane 2) and 3 (lane 3) for fkbp4 (panel B) and clones 3 (lane 3), 6 (lane 6), 7 (lane 7), and 10 (lane10) for fkbp5 (panel C) showed plasmid bands with molecular weight higher than that of the control plasmid pBAC-FB. This result indicates that gene retrieval by gap repair occurred in these clones. More colonies were analyzed to see the gene retrieval efficiency for each of the three genes (Table 2). The retrieval efficiency of kiss1r was 12.2 % which is lower than that of the other two genes, fkbp4 and fkbp5. It is not known that gene retrieval efficiency is affected by gene toxicity of kiss1r, even in the low-copy-number vector. The overall retrieval efficiency of all three genes was 23 %. Successful retrieval of the sequences was also confirmed by restriction analysis of the plasmids (pBAC-FB-kiss1r, pBAC-FB-fkbp5, and pBAC-FB-fkbp4) and, finally, by nucleotide sequencing of the border region of the cloned inserts (sequence data not shown). NheI digestion of pBAC-FB-kiss1r produced the expected fragments: 13.71, 3.06, 1.82 kb (Fig 3b lane 1). XbaII digestion of pBAC-FB-51 produced the expected fragments: 8.62, 6.55, 1.77, 0.92 kb (Fig. 3b lane 2). Xba1 digestion of pBAC-FB-52 produced the expected fragments: 6.55, 3.48, 3.39, 2.85, 1.25, 0.27 kb (Fig. 3b lane 3).

Fig. 2.

Fig. 2

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Colony cracking analysis of gene retrieval from BACs containing kiss1r (a), fkbp4 (b), and fkbp5 (c). Positions of fragmented chromosome, recombinant plasmid with a retrieved gene, and the non-recombinant control plasmid pBAC-FB (lane M) were indicated by arrows with letters “a,” “b,” and “c,” respectively. Some colonies show no visible plasmid band (see “Discussion” section)

Table 2.

Efficiency of gene retrieval by gap repair with pBAC3.6-FB plasmid

Target
gene		 No. of colonies
tested		 No. of colonies
with gene retrieval		 % colonies with
gene retrieval
kiss1r 49   6 12.2
fkbp4 40 10 25.0
fkbp5 40 14 35.0
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Fig. 3.

Fig. 3

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Linear maps of pBAC-FB-kiss1r, pBAC-FB-fkbp4, pBAC-FB-fkbp5, and pBAC-FB-fkbp4-FNFL (a) and restriction analysis of the plasmids (b, c). Exons were numbered. pBAC-FB-fkbp4-FNFL contained exon 2 flanked by two loxPs (filled triangles). Neo (positive selection marker, open box) flanked by two FRTs (open triangles) was inserted in intron 2. The recognition sites for restriction endonucleases are indicated with lines with letters: N NotI, Nh NheI, X XbaI, B BamHI. pBAC-FB-kiss1r was digested with NheI (panel b, lane 1). pBAC-FB-fkbp4 and pBAC-FB-fkbp5 were digested with XbaI (panel B, lane 2 and 3, respectively). pBAC-FB-fkbp4-FNFL was digested with BamHI (panel c, lane 1). Low-molecular-weight DNA fragments are barely visible

Construction of a Conditional Targeting Vector Using a Retrieved Genomic DNA

The application of pBAC-FB for constructing a conditional targeting vector was tested using the retrieved genomic region containing the fkbp4 gene as a target. The first step was to insert the LNL cassette of pL452 into the intron 1 (upstream of exon 2) of the fkbp4 gene cloned in pBAC-FB-fkbp4. The insertion of the LNL cassette increased the plasmid size from 17.7 to 19.7 kb. The size difference was identified by colony cracking analysis (data not shown). The insertion was also confirmed by gel electrophoresis of purified plasmids and, finally, by nucleotide sequence determination (sequence data not shown). The next step was to delete the inserted LNL cassette leaving only a loxP site upstream of exon 2 by arabinose-induced Cre expression. The deletion was confirmed by colony cracking analysis and gel electrophoresis of purified plasmids (data not shown). The final step was to insert the FNFL cassette of pL451 in intron 2 (just downstream of exon 2) by homologous recombination. The final conditional targeting construct was confirmed by colony cracking analysis (data not shown). The locations of the loxP and the FNFL cassette were confirmed by restriction analysis (Fig. 3c) and nucleotide sequence determination (sequence data not shown). We also successfully constructed a conditional targeting vector for the kiss1r gene from pBAC-FB-kiss1r (data not shown). Generation of a conditional knockout mouse for kiss1r is in progress (and will be described elsewhere).

Incompatibility Between BAC and the Low-Copy-Number Retrieval Plasmids

The low-copy-number plasmid pBAC-FB was derived from pBACe3.6, which was originated from the F plasmid and was used as a vehicle for the mouse BAC libraries. Because BAC and the retrieval plasmids pBAC-FB and pBAC-FC share the same replication origin, they would compete with each other when they co-exist in a host cell. These vectors are stringently maintained at one to two copies in a cell [20]. BAC and the introduced retrieval plasmid may co-exist and compete with each other after successful gene retrieval by homologous recombination. We tested the incompatibility of these two plasmids by introducing pBAC-FB into an E. coli host carrying the BAC containing fkbp5 gene. After selecting transformed cells on an LB+Amp plate for 24 h, single colonies were resuspended in 1 ml of LB broth and serially diluted. Antibiotic-resistant cells in single colonies were counted by spreading the serially diluted cell suspension onto LB+Amp plates and LB+Cam plates. Among 10 colonies tested, no colonies appeared on the LB+Cam plates. We also streaked single colonies on LB+Cam plates, but again no colonies appeared, suggesting that the targeting vector pBAC-FB completely excluded the endogenous BAC plasmid under selection pressure against BAC in the presence of Amp. This complete exclusion might be due to the nature of having large sized DNA sequence (300 kb) of the BAC clone, which would be a disadvantage for competition with the much smaller plasmid pBAC-FB. When pBAC-DE, which is smaller than pBAC-FB, was used instead of BAC, however, the results were the same, suggesting that the size was not the cause for the complete exclusion. This does not, however, mean that plasmid size has nothing to do with competition between two plasmids of different sizes. In order to test the effects of plasmid size on competition between two compatible plasmids, we introduced the high-copy-number plasmid pL253 which was derived from pBluescript and does not belong to the same incompatibility group as the BAC plasmid. E. coli that carried BAC or pBAC-DE was also analyzed. The high-copy-number plasmid pL253 effectively competed out the BAC under selection pressure, allowing only a small fraction of the colonies to carry both plasmids (Table 3). pL253 did not exert any effects on the replication of a much smaller plasmid, pBAC-DE, which shares the same replication machinery as that for BAC. These results suggest that plasmid size may have a high competition effect between two compatible plasmids if their size differences are extreme.

Table 3.

Incompatibility test of plasmids

Incoming
plasmid		 Resident
plasmid		 No. of cell
doublings		 % cells carrying resident
plasmid in a single colony
pL253 BACa 22.9 ± 0.5b 13.4 ± 5b
pL253 pBAC-DE 23.1 ± 0.4c 103 ± 7c
pBAC-FC BACa 25.0 ± 0.6c 0c
pBAC-FC pBAC-DE 24.0 ± 0.4c 0c
pBAC-DE pBAC-FC 24.6 ± 0.5c 0c
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Antibiotic selection was applied against resident plasmids

a

The size of the BAC clone (PR23-23B10) was 228 kb

b

The number of colonies tested is 5

c

The number of colonies tested is 10

Gene Targeting in Embryonic Stem Cells

After 12 days of selection, 45 G418-resistant embryonic stem cell colonies were picked and expanded. Eleven out of the forty-five G418-resistant cell clones showed the products of the expected size, 2,114 base pairs by PCR amplification (Fig. 4). The PCR products from the ten clones were cloned to determine their nucleotide sequences. All of them had correctly recombined fkbp4 sequences (sequence data not shown). Homologous recombination at the long arm region is underway.

Fig. 4.

Fig. 4

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Targeting embryogenic stem cells with the low-copy-number targeting vector, pBAC-FB-fkbp4-FNFL. a Schematic drawing of a targeted fkbp4 allele is shown. Positions of PCR primers for PCR amplification of one end of the targeted allele are indicated by arrows. b PCR-amplified products from genomic DNAs of 45 G418-resistant embryonic stem cell clones (1–45) and non-transfected embryonic stem cells (C) were separated along with molecular size markers (M) on 1.5 % agarose gels. Arrows indicate positive DNA bands with a size of 2,114 base pairs (bp)

Discussion

Recombineering using λ Red recombinase-mediated homologous recombination was introduced as an efficient way of constructing a conditional targeting vector by combining homologous recombination system with site-specific recombination systems, such as the Cre-loxP or Flp-FRT systems [3, 21].

Construction of a targeting vector using recombineering is usually performed in a high-copy-number vector, because high-copy-number vectors can be easily purified in a sufficient amount for a variety of applications, such as gene manipulation and transfection experiments. However, cloning and subsequent gene manipulation of cloned DNA in a high-copy-number vector may not be possible for toxic genomic sequences due to cell death caused by the high level of gene dosage effects of the toxic sequences. In order to clone toxic genes into high-copy-number vectors, the expression of the toxic genes must be tightly regulated [22]. The tight regulation is not applicable to genomic sequences in which the position or identity of the toxic sequence is not known. Furthermore, for a genomic fragment that is used to construct a targeting vector, any extra manipulation for tight regulation, except the changes needed for gene knockout or knock-in, is not desirable. Another method is to clone the toxic sequences into a low-copy-number vector to reduce gene dosage effects [23]. The latter is an approach that is more applicable to potentially toxic genomic fragments in which toxic sequences are not identified.

Cloning vectors with low-copy-number have been reported in other studies. Vectors containing the replication origin (oriF) of F plasmid have been developed to control the expression levels of cloned genes or to avoid the toxicity of cloned genes [20, 24]. Vectors with six to eight copies per cell have been developed for a wide range of applications such as cloning toxic genes, complementation analysis, unidirectional deletions of cloned genes, and so on [25]. In this study, the cloning vector pBAC-FB which contains the replication origin (oriF) of F plasmid was constructed and tested for retrieving potential toxic genomic genes from BAC. It was also tested for construction of conditional targeting vectors by recombineering which utilizes phage λ Red recombinase and the cre-loxP system, and for embryonic stem cell targeting.

We tried to retrieve the kiss1r gene from a BAC clone using the high-copy-number vector pL253 through Red-mediated homologous recombination in E. coli SW106. However, because of unknown reasons, the retrieval was unsuccessful. We suspect that this failure was the result of an unidentified toxic sequence in the DNA which is to be retrieved. Although it has been reported that the kiss1r gene was cloned and that this gene was used to construct a conventional (not conditional) knockout targeting vector, the region cloned was different from that cloned in this study, in that the region cloned contained a 5′ promoter region, part of exon 1, exon 2, and intron 2 [26], whereas the cloned region of kiss1r in this study contains all of the exons and the introns of kiss1r. The low-copy-number plasmid pBAC-FB was used successfully in retrieving kiss1r gene fragment from BAC. The same end homology sequences that were used to retrieve kiss1r in pL253 were used to retrieve kiss1r from pBAC-FB. This result suggests that a potentially toxic sequence is contained in the kiss1r gene.

pBAC-FB was also used to retrieve other DNA fragments, including parts of the fkbp4 or fkbp5 genes, from BAC clones, suggesting that BAC clones can be a substitution for high-copy-number vectors when DNA retrieval is unsuccessful. Furthermore, the retrieved DNA fragments, parts of kiss1r or fkbp4 in pBAC-FB, were manipulated without any difficulty using Red-mediated homologous recombination and the Cre-loxP system to generate conditional knockout targeting vectors for these genes.

There are, however, several potential disadvantages in using pBAC-FB. One of these disadvantages is in the large size of the pBAC-FB vector. Some investigators prefer to use a PCR-amplified plasmid as a retrieval vector, using synthetic primers with end homology sequences (45 bp or longer homology) instead of cloning PCR-amplified end homologies (200–500 bp) in the vector and making the plasmid linear by cutting at an intentionally generated restriction site between end homologies. It is difficult to amplify the whole plasmid when investigators use this method, because of the large size of the plasmid. The other disadvantage is in its low yield in plasmid isolation. For colony cracking analysis on agarose gel, a number of cells equivalent to three to four colonies, instead of one colony for a high-copy-number plasmid, should be used to see a plasmid band on agarose gels. One-fifth of a mini-scale plasmid preparation (mini-preparation), instead of one-fiftieth for a high-copy-number plasmid, from a 1.5 ml culture was used to see bands of non-restricted or restricted plasmid on agarose gels. For nucleotide sequence determination, three or four mini-scale plasmid preparations that are combined and concentrated by ethanol precipitation can be routinely used.

The overall gene retrieval efficiency of the low-copy-number vector pBAC-FB is 23 %. Using the same gene retrieval procedure, more than 90 % of efficiency was obtained by the high-copy-number vector pL253 (data not shown). In addition to the low retrieval efficiency, some colonies did not show any plasmid bands in colony cracking analysis (Fig. 2). These colonies were thought to have the retrieval plasmid integrated into BAC or the host chromosome. Integration into BAC was more likely to happen, because there was a long stretch of homology (6.4 kb), including the F plasmid replicon and chloramphenicol resistance, between pBAC-FB and BACs. Chromosome integration can be another possibility because integration of linear and circular plasmids into chromosome has been reported in Bacillus subtilis [27] and E. coli [28]. These integration events in BAC or chromosome can interfere with gene retrieval and reduce the efficiency.

For high-copy-number vectors, recombinants and non-recombinants co-exist in a cell [29]. Therefore, we need to purify plasmids (mixture) and re-introduce them into E. coli host cells to select the cells that carry the correctly engineered plasmid on an LB plate supplemented with the appropriate antibiotic. An advantage in using pBAC-FB is that it completely eliminates any residual BAC plasmid in E. coli after DNA retrieval on selective medium.

The reason for this complete elimination is that the replication of BAC and pBAC-FB is tightly regulated by chromosome replication. Their copy number is maintained at only one to two copies in a cell. If BAC exists at 1 copy in a cell, it is linearized by losing the gene of interest as the incoming linear pBAC-FB gets the gene from it by gap repair and is circularized. The linearized BAC is not retained as a plasmid in the cell. If BAC exists at 2 copies in a cell, a residual circular BAC and pBAC-FB with a retrieved gene co-exist in a cell after gene retrieval. The two plasmids segregate as the cell divides. Cells with pBAC-FB with a retrieved gene survive while cells with BAC only are eliminated in the presence of ampicillin in the medium. pBAC-FB also eliminates any non-recombinant plasmid, if any, when an LNL cassette or an FNFL cassette is introduced in the retrieved DNA on selective medium in the process of a conditional targeting vector construction.

The replication of the low-copy-number plasmid pBAC-FB is as tightly regulated as that of a BAC. This plasmid can be used to clone and manipulate potentially toxic genomic DNA sequences. Theoretically, it is possible to clone in pBAC-FB any sequence from a BAC, because the two plasmids are maintained at the same copy number (one to two copies per cell) in E. coli and provide the same gene dosage to the host. The newly developed pBAC-FB vectors, therefore, offer a solution for cloning sequences that are potentially toxic and an improved subcloning tool for generating gene-targeting vectors.

The low-copy-number targeting vector, pBAC-FB-fkbp4-FNFL, was tested for embryonic stem cell targeting at its short arm homology region. Targeting efficiency was 4.5 × 10−5. The ratio was within the efficiency range (10−5–10−6) reported in high-copy-number targeting vectors [30]. The results show that the low-copy-number targeting vector can be used to target embryonic stem cells with efficiency comparable to that of high-copy-number targeting vectors.

Acknowledgments

This work was supported by a grant from Inje Research and Scholarship Foundation in 2012, by a grant from Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2011-0011173), and by a grant UO1 HD066432 from National Institutes of Health.

Abbreviations

Amp

Ampicillin

AmpR

Ampicillin resistance

BAC

Bacterial artificial chromosome

Cam

Chloramphenicol

CamR

Chloramphenicol resistance

FNFL

FRT-Neo-FRT-loxP

Kan

Kanamycin

LB

Luria-Bertani medium

LB+Amp

LB medium containing ampicillin

LB+Cam

LB medium containing chloramphenicol

LB+Kan

LB medium containing kanamycin

LNL

loxP-Neo-loxP

PCR

Polymerase chain reaction

Contributor Information

Giyoun Na, School of Biological Sciences, Inje University, Gimhae 621-749, South Korea.

Andrew Wolfe, Division of Endocrinology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

CheMyong Ko, Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 3806 VMSBS, MC-002, 2001 South Lincoln Avenue, Urbana, IL 61802, USA.

Hyesook Youn, Department of Bioscience and Biotechnology, Sejong University, Kwangjin gu, Seoul 143-747, South Korea.

Young-Min Lee, Department of Microbiology, College of Medicine, Chungbuk National University, Cheongju, Chungbuk 361-763, South Korea; Department of Animal, Dairy, and Veterinary Sciences, Utah Science Technology and Research (USTAR), College of Agriculture, Utah State University, Logan, UT 84322-4815, USA.

Sung June Byun, Animal Biotechnology Division, National Institute of Animal Science, Suwon 441-706, South Korea.

Iksoo Jeon, Research Planning Team, National Institute of Animal Science, Suwon 441-706, South Korea.

Yongbum Koo, Email: mbkooyb@inje.ac.kr, School of Biological Sciences, Inje University, Gimhae 621-749, South Korea.

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