- Technical Note
- Open Access
A set of vectors for introduction of antibiotic resistance genes by in vitro Cre-mediated recombination
© Vassetzky et al; licensee BioMed Central Ltd. 2008
- Received: 19 November 2008
- Accepted: 23 December 2008
- Published: 23 December 2008
Introduction of new antibiotic resistance genes in the plasmids of interest is a frequent task in molecular cloning practice. Classical approaches involving digestion with restriction endonucleases and ligation are time-consuming.
We have created a set of insertion vectors (pINS) carrying genes that provide resistance to various antibiotics (puromycin, blasticidin and G418) and containing a loxP site. Each vector (pINS-Puro, pINS-Blast or pINS-Neo) contains either a chloramphenicol or a kanamycin resistance gene and is unable to replicate in most E. coli strains as it contains a conditional R6Kγ replication origin. Introduction of the antibiotic resistance genes into the vector of interest is achieved by Cre-mediated recombination between the replication-incompetent pINS and a replication-competent target vector. The recombination mix is then transformed into E. coli and selected by the resistance marker (kanamycin or chloramphenicol) present in pINS, which allows to recover the recombinant plasmids with 100% efficiency.
Here we propose a simple strategy that allows to introduce various antibiotic-resistance genes into any plasmid containing a replication origin, an ampicillin resistance gene and a loxP site.
- Antibiotic Resistance Gene
- Target Vector
- LoxP Site
- Chloramphenicol Acetyl Transferase
Antibiotics blasticidin S, puromycin and G418 are frequently used for selection of stably transfected mammalian cell lines . For this purpose plasmid expressing a gene of interest may be cotransfected with a plasmid containing a convenient antibiotic resistance gene [2, 3]. Alternatively the antibiotic resistance gene and the gene of interest can be combined in one plasmid .
Unfortunately, the choice of several antibiotic resistance markers is available only for few types of expression vectors (for example, pcDNA3.1 vector series, Invitrogen) . That is why usually researchers have to introduce new antibiotic resistance genes into the original vector. In this case, the cloning strategy may be complicated by the absence of the unique and convenient restriction sites in the plasmids containing long inserts.
Construction of the insertion vectors pINS
We have used the backbone of the pUNI-10 plasmid [12, 13] (Fig 1, 2) for construction of the insertion vectors pINS-Puro, pINS-Neo and pINS-Blast. pUNI-10 contains the R6Kγ origin of replication [14, 15] and the LoxP site  recognized by Cre recombinase . R6Kγ origin is active only in E. coli strains expressing the π-protein encoded by the pir gene. Cloning and production of the pINS plasmids was performed in the pir+ E. coli strain BW23474 expressing the mutant form of the π-protein (pir-116) that allows to maintain a plasmid with the R6Kγ origin at a high copy number [17, 13].
Thus the pINS vectors contain four principal elements:
- R6Kγ origin of replication;
- LoxP site required for Cre-mediated recombination with target vector;
- Genes coding for either chloramphenicol acetyl transferase  or aminophosphotransferase  providing the resistance to the antibiotics chloramphenicol (Cam) or kanamycin (Kan), respectively. These genes are required for the selection of the recombinant constructs in E. coli;
- Genes coding for either pac (puromycin-N-acetyl transferase), aph (aminoglycoside phosphotransferase) or bsd (blasticidin S deaminase) controlled by the SV40 promoter. These genes provide mammalian cells with the resistance to puromycin, G418, or blasticidin S.
Conventional E. coli strains (XL-1 Blue, DH5α, JM-109 etc.) are pir- and cannot maintain the pINS plasmid. In contrast, the products of in vitro recombination between the pINS plasmid and the target vector can successfully replicate in the pir- strains due to the presence of the active origin of replication provided by the target vector. The selection of the recombinant plasmids is achieved by the markers Kan or Cam provided by pINS plasmid. This selection procedure allows to achieve 100% yield of recombinant plasmids (Fig. 1).
Construction of the target vectors
Target vectors compatible with our pINS plasmids must contain only three necessary elements (Fig. 1):
- the LoxP site;
- An origin of replication active in the pir- E. coli strain, for example, pUC-origin ;
- An appropriate antibiotic resistance gene, for example beta-lactamase (bla)  providing resistance to ampicillin (Amp).
We have modified several commercially available plasmids (phRL-TK (Promega) and pBluescriptII (Stratagene) by introduction of the LoxP sites resulting in the target vectors pT-TK and pT-BS respectively (Fig 2).
pT-TK vector contains the Renilla luciferase gene under control of the herpes simplex virus thymidine kinase promoter (TK) . pT-TK vector can be used for the expression of a gene of interest at the levels that are 10-20 times lower than produced by the CMV promoter at least in some types of mammalian cells (HeLa, NIH 3T3  and MEF ). For this purpose, the luciferase gene has to be cut out by NheI and XbaI and replaced by the gene of interest. Alternatively, any other vector can be used as a target vector in our system if upgraded by insertion of the LoxP sites as described .
pT-BS vector contains the convenient pBluescriptII polylinker  suitable for cloning of the expression modules containing a gene of interest under the control of appropriate promoter.
We have also used the commercially available target vector phrGFP (Stratagene) already containing the LoxP site. We have also created a pT-FLAG vector by replacing the GFP via FLAG-tag in the phrGFP vector (Fig 2).
All target vectors were cloned and produced in the XL-1 Blue strain (pir-).
Introduction of an antibiotic resistance gene in the target vectors by in vitro recombination
We have performed in vitro recombination between the pINS and the target vectors using Cre-recombinase. We have transformed the pir- and pir+ E. coli strains (XL-1 Blue and BW23474 respectively) with the reaction mixture in order to test the efficiency of the reaction and selected the transformants using either kanamycin, chloramphenicol or ampicillin.
In order to test the purity of our plasmid preparations, we have also transformed the pINS vectors and the target vectors into both pir- and pir+ strains and selected the transformants using either kanamycin/chloramphenicol or ampicillin (Fig. 3 and data not shown). As expected, the pINS vectors did not transform the pir- strain. In contrast, the pir+ strain transformed by the pINS vector can grow on either kanamycin or chloramphenicol, but not on ampicillin. The target vector transformed both XL1-Blue (pir-) and BW23474 (pir+) strains since the activity of the pUC origin of replication did not depend on the presence of the pir gene and produced the ampicillin-resistant, but neither kanamycin- nor chloramphenicol-resistant colonies. This confirmed the purity of the initial plasmids.
We calculated the yield of recombination (0.02%) by counting the kanamycin-resistant colonies of the pir- strain transformed by the recombination mix and taking into account the transformation efficiency (2.2 × 10^8 colonies/mkg DNA) (Fig. 3, and data not shown).
Researchers working with the vectors suitable for expression in mammalian cells often meet with the puzzle of how to quickly introduce or switch the antibiotic resistance gene in the vector of interest. In this paper we describe the pINS vectors suitable for introduction of antibiotic resistance genes in any plasmid of interest containing the LoxP site by Cre-mediated recombination. We cloned the genes providing resistance to three frequently used antibiotics (puromycin, blasticidin S and G418) in the pUNI-10 plasmid . The plasmid pUNI-10 contains the R6Kγ origin of replication which is inactive in majority of E. coli strains routinely used for cloning (they are pir-). The plasmid resulting from recombination between the pINS and a target vectors acquires a gene of interest (provided by the target vector) and resistance to an antibiotic (puromycin, blasticidin S or G418) (provided by pINS vector). The principle of selection in based on the acquisition of the functional origin of replication (provided by the target vector) as well as the marker providing resistance to kanamycin or chloramphenicol (provided by the pINS vector). Combination of these features makes it possible to select the recombinant plasmids by transforming the recombination mix into a pir- E. coli strain and selecting the kanamycin- or chloramphenicol-positive colonies (Fig. 1). These two features are necessary and sufficient for selection of the recombinant plasmids. Utilization of two antibiotics (chloraphenicol/kanamycin+ampicillin) does not provide any further enhancement to the procedure.
Our method is similar to the procedure used in the existing pExchanger system (Stratagene). pExchanger system relies on integration of the linear fragments coding for the antibiotic resistance genes and selection marker (Kan or Cam) into the target vector using Cre-recombination. Linear fragments can't replicate by themselves and thus can't efficiently transform E. coli. Presumably only the colonies containing the recombinant plasmids can be selected by the markers encoded in the linear fragments. In contrast to pExchanger system, we propose to use the replication-deficient circular pINS plasmids that can be easily produced in the user laboratory and thus allow the user to cut the cost of the cloning.
The Insertion vectors pINS-Puro, pINS-Neo and pINS-Blast are compatible with the numerous target vectors already present on the market (for example, pExchange core or phrGFP vector families, Stratagene). In addition, virtually any vector of interest can be converted into a target vector by simple introduction of the LoxP site.
In order to obtain the pINS-Blast vector, the pcDNA6/TR vector (Invitrogen, Carlsbad, CA, USA) was digested by XmnI and SalI and the fragment containing blasticidin S resistance gene was ligated with the pUNI-10 vector (kindly provided by Dr. Stephen Elledge) digested by EcoRV and SalI (these and other restriction enzymes were purchased from Fermentas, Vilnius, Lithuania) using T4 ligase (Fermentas). The product of ligation was transformed by electroporation into the strain BW23474 (Δlac-169 rpoS(Am) robA1 creC510 hsdR514 endA recA1 uidA (ΔMluI)::pir-116) that was kindly provided by Dr. Stephen Elledge, and selected using kanamycin. The resulting plasmid was named pINS-Blast.
In order to obtain the pINS-Puro vector, the pPur plasmid (Clontech, Terra Bella, CA, USA) was digested by NdeI, blunt-ended using Klenow fragment (Fermentas), then digested by BamHI and ligated with the fragment of pACYC-184 plasmid  containing Cam resistance gene (pACYC-184 was purchased from Fermentas). In order to obtain this fragment we first digested pACYC-184 by BclI, treated it with Klenow fragment, then digested with BamHI. The product of ligation was named pACYC-Puro. pACYC-Puro was digested by Bst1107I, BamHI, then blunt-ended with Klenow fragment. The fragment containing chloramphenicol and puromycin resistance genes was ligated with pUNI-10 vector digested EcoRI and BglII and blunt-ended with Klenow fragment. The product of ligation was transformed via electroporation in BW23474 strain and selected via chloramphenicol. The resulting plasmid was named pINS-Puro.
In order to obtain the pINS-Neo vector, pCINeo plasmid (Promega, Madison, WI, USA) was digested by BamHI and the fragment containing pUC-origin, ampicillin and neomycin resistance genes was ligated to BamHI-linearized pUNI-10 resulting in the plasmid "3490". Plasmid "3490" was digested by XbaI and self-ligated in order to remove ampicillin resistance gene and pUC-origin of replication. The product of ligation was transformed into the BW23474 strain via electroporation and selected using kanamycin. The resulting plasmid was named pINS-Neo.
In order to prepare the pT-FLAG vector, we digested phrGFP vector (Stratagene) by NheI and EcoRI and replaced the GFP ORF by the oligonucleotide duplex encoding the FLAG-tag (oligo1: 5'-CTAGCCCATGGATTACAAAGACGATGACGATAAACCTAGCTTCG; oligo2: 5'-AATTCGAAGCTAGGTTTATCGTCATCGTCTTTGTAATCCATGGG) (Sigma, St. Louis, MO, USA). Then LoxP-site coupled to ampicillin resistance gene was isolated from pT-FLAG plasmid by BspHI digestion and cloned into phRL-TK (Promega, Madison, WI, USA) or pBlueScriptII (Stratagene) digested by BspHI. The resulting plasmids were named pT-TK and pT-BS respectively.
In vitro Cre recombination
500 ng of pINS vector was mixed with 500 ng of target vector, 5 units of Cre recombinase (Stratagene, La Jolla, CA, USA), and a buffer recommended by manufacturer in 10 mkl reaction volume. The reaction was incubated for 30 min at 37°C, then Cre recombinase was heat-inactivated at 65°C for 20 min. The recombination mix was transformed by electroporation into XL1-Blue E. coli strain (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F' proAB lacI qZ ΔM15 Tn10 (Tetr)]) purchased from Stratagene and selected using either 34 mkg/ml chloramphenicol (Euromedex) or 50 mkg/ml kanamycin (Euromedex, Mundolsheim, France).
Testing the performance of the constructs in HeLa
Dulbecco's modified Eagle's medium (DMEM #31885 Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated Millerium Fetal Bovine Serum (#BWSTS1810 VWR International, Fontenay-sous-Bois, France), 100 units/ml penicillin G and 100 mkg/ml streptomycin sulfate (Gibco) and 500 ng/ml Fungizone (Invitrogen) in the presence of 5% CO2. Cells were transfected by 1 mkg of plasmid DNA using JetPEI (Polyplus-transfection Inc., New York, NY, USA) according to the protocol provided by supplier. 24 hours after transfection media was replaced and the cells were either grown for another 24 hours (transient transfection) or selected with either 3 mg/ml puromycin (Sigma #P8833) for 3 days, 5 mg/ml blasticidin S (Sigma #15205) for 5 days or 1 mg/ml G418 (Sigma #G8168) for 10 days until the complete death of mock-transfected cells. Transfected cells were either stained by 1% methylene blue (Euromedex #A514) in 50% methanol or mounted on the slides, stained by 100 ng/ml DAPI (Sigma #D9542) and inspected under the fluorescent microscope Olympus AX70.
Plasmids and their sequences are available upon request.
Authors are grateful to Andrei Petrov for his criticism, ideas and support. This research has been supported by grants from the Institut National de Cancer (INCa), the Association Française contre les Myopathies (AFM) and the Fondation de France.
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