Limits...
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

pINS plasmids are introduced always in the same orientation. Upper part. Schematic representation of the Cre-mediated recombination between Insertion vector pINS-Puro and Target vector phrGFP. Recombination intermediate and the sizes of the plasmids are shown. Lower part. Orientation of the pINS-Puro insert in the product of recombination (Puro × GFP) was analyzed by EcoRI. The sizes of the digestion products were analyzed on the agarose gel. We observed the fragments specific to only one orientation of the pINS-Puro in the product of recombination. This orientation is determined by the orientation of loxP sites.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2654900&req=5

Figure 4: pINS plasmids are introduced always in the same orientation. Upper part. Schematic representation of the Cre-mediated recombination between Insertion vector pINS-Puro and Target vector phrGFP. Recombination intermediate and the sizes of the plasmids are shown. Lower part. Orientation of the pINS-Puro insert in the product of recombination (Puro × GFP) was analyzed by EcoRI. The sizes of the digestion products were analyzed on the agarose gel. We observed the fragments specific to only one orientation of the pINS-Puro in the product of recombination. This orientation is determined by the orientation of loxP sites.

Mentions: In order to test the integrity of the recombination product, we have picked either kanamycin- or chloramphenicol-resistant colonies, isolated plasmid DNA and digested it with an appropriate restriction enzyme. We used EcoRI in case of recombination between pINS-Puro and phrGFP. All colonies gave the restriction pattern expected for the product of recombination, thus efficiency of the resistance marker introduction is close to 100% (Fig 4 and data not shown). Moreover, due to the directional nature of the LoxP sites, integration occurs in only one orientation depending on the orientation of the LoxP sites. This feature makes the population of recombinant vectors highly homogenous (Fig. 4 and data not shown).


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

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

pINS plasmids are introduced always in the same orientation. Upper part. Schematic representation of the Cre-mediated recombination between Insertion vector pINS-Puro and Target vector phrGFP. Recombination intermediate and the sizes of the plasmids are shown. Lower part. Orientation of the pINS-Puro insert in the product of recombination (Puro × GFP) was analyzed by EcoRI. The sizes of the digestion products were analyzed on the agarose gel. We observed the fragments specific to only one orientation of the pINS-Puro in the product of recombination. This orientation is determined by the orientation of loxP sites.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: pINS plasmids are introduced always in the same orientation. Upper part. Schematic representation of the Cre-mediated recombination between Insertion vector pINS-Puro and Target vector phrGFP. Recombination intermediate and the sizes of the plasmids are shown. Lower part. Orientation of the pINS-Puro insert in the product of recombination (Puro × GFP) was analyzed by EcoRI. The sizes of the digestion products were analyzed on the agarose gel. We observed the fragments specific to only one orientation of the pINS-Puro in the product of recombination. This orientation is determined by the orientation of loxP sites.
Mentions: In order to test the integrity of the recombination product, we have picked either kanamycin- or chloramphenicol-resistant colonies, isolated plasmid DNA and digested it with an appropriate restriction enzyme. We used EcoRI in case of recombination between pINS-Puro and phrGFP. All colonies gave the restriction pattern expected for the product of recombination, thus efficiency of the resistance marker introduction is close to 100% (Fig 4 and data not shown). Moreover, due to the directional nature of the LoxP sites, integration occurs in only one orientation depending on the orientation of the LoxP sites. This feature makes the population of recombinant vectors highly homogenous (Fig. 4 and data not shown).

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