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A set of vectors for introduction of antibiotic resistance genes by in vitro Cre-mediated recombination.

Dmitriev PV, Vassetzky YS - BMC Res Notes (2008)

Bottom Line: 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 R6Kgamma replication origin.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Université Paris-Sud 11 CNRS UMR 8126 < Interactions moléculaires et cancer >, Institut de Cancérologie Gustave-Roussy, F-94805 Villejuif cedex, France. vassetzky@igr.fr.

ABSTRACT

Background: 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.

Findings: 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 R6Kgamma 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Maps of Target (phRGFP, pT-FLAG, pT-TK and pT-BS) and Insertion (pINS-Blast, pINS-Puro and pINS-Neo) vectors. pUNI-10 vector used as a backbone for cloning of the antibiotic resistance genes (Blast, Puro and Neo) is also shown. Only relevant restriction sites are shown. Detailed maps are available upon request.
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Figure 2: Maps of Target (phRGFP, pT-FLAG, pT-TK and pT-BS) and Insertion (pINS-Blast, pINS-Puro and pINS-Neo) vectors. pUNI-10 vector used as a backbone for cloning of the antibiotic resistance genes (Blast, Puro and Neo) is also shown. Only relevant restriction sites are shown. Detailed maps are available upon request.

Mentions: Here we propose to introduce the antibiotic resistance genes using recombination (Fig. 1). We have created several insertion vectors (pINS-Puro, pINS-Neo, pINS-Blast) containing the pac (puromycin-N-acetyl transferase) [6,7], aph (aminoglycoside phosphotransferase) [8,9] and bsd (blasticidin S deaminase) [10] genes that provide resistance to puromycin, G418 (G418 is an aminoglycoside, similar in structure to neomycin) and blasticidin S respectively (Fig 2). pINS vectors can be introduced via Cre-recombination [11] into several commercially available target vectors containing the LoxP sites, for example phrGFP vector (Stratagene). In addition we created several new target vectors: pT-FLAG, pT-BS and pT-TK (Fig. 2).


A set of vectors for introduction of antibiotic resistance genes by in vitro Cre-mediated recombination.

Dmitriev PV, Vassetzky YS - BMC Res Notes (2008)

Maps of Target (phRGFP, pT-FLAG, pT-TK and pT-BS) and Insertion (pINS-Blast, pINS-Puro and pINS-Neo) vectors. pUNI-10 vector used as a backbone for cloning of the antibiotic resistance genes (Blast, Puro and Neo) is also shown. Only relevant restriction sites are shown. Detailed maps are available upon request.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654900&req=5

Figure 2: Maps of Target (phRGFP, pT-FLAG, pT-TK and pT-BS) and Insertion (pINS-Blast, pINS-Puro and pINS-Neo) vectors. pUNI-10 vector used as a backbone for cloning of the antibiotic resistance genes (Blast, Puro and Neo) is also shown. Only relevant restriction sites are shown. Detailed maps are available upon request.
Mentions: Here we propose to introduce the antibiotic resistance genes using recombination (Fig. 1). We have created several insertion vectors (pINS-Puro, pINS-Neo, pINS-Blast) containing the pac (puromycin-N-acetyl transferase) [6,7], aph (aminoglycoside phosphotransferase) [8,9] and bsd (blasticidin S deaminase) [10] genes that provide resistance to puromycin, G418 (G418 is an aminoglycoside, similar in structure to neomycin) and blasticidin S respectively (Fig 2). pINS vectors can be introduced via Cre-recombination [11] into several commercially available target vectors containing the LoxP sites, for example phrGFP vector (Stratagene). In addition we created several new target vectors: pT-FLAG, pT-BS and pT-TK (Fig. 2).

Bottom Line: 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 R6Kgamma replication origin.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Université Paris-Sud 11 CNRS UMR 8126 < Interactions moléculaires et cancer >, Institut de Cancérologie Gustave-Roussy, F-94805 Villejuif cedex, France. vassetzky@igr.fr.

ABSTRACT

Background: 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.

Findings: 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 R6Kgamma 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus