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

Functionality of GFP gene from phRGFP plasmid is preserved in the products of recombination with pINS-Puro, pINS-Neo or pINS-Blast plasmids. HeLa cells transfected by 1 mkg of either Target vector phrGFP or the products of recombination (Blast × GFP, Neo × GFP and Puro × GFP). Cells transfected by the recombination products were selected by either blasticidin S, G418 or puromycin and stained by DAPI. Expression of the GFP was analyzed under the microscope. In case of transient transfection by phrGFP vector we usually observed 25% GFP positive cells. In contrast we observed that 80–100% of the cells transfected by the products of recombination and selected by the corresponding antibiotics are GFP positive. Scale bar: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Functionality of GFP gene from phRGFP plasmid is preserved in the products of recombination with pINS-Puro, pINS-Neo or pINS-Blast plasmids. HeLa cells transfected by 1 mkg of either Target vector phrGFP or the products of recombination (Blast × GFP, Neo × GFP and Puro × GFP). Cells transfected by the recombination products were selected by either blasticidin S, G418 or puromycin and stained by DAPI. Expression of the GFP was analyzed under the microscope. In case of transient transfection by phrGFP vector we usually observed 25% GFP positive cells. In contrast we observed that 80–100% of the cells transfected by the products of recombination and selected by the corresponding antibiotics are GFP positive. Scale bar: 20 μm.

Mentions: Then HeLa cells resistant to the antibiotics were inspected under the microscope for the expression of GFP. Only cells transfected by the recombination products were GFP-positive. Moreover, the proportion of the GFP-positive cells was considerably higher than in the case of transient transfection by the phrGFP plasmid (Fig 6). We conclude that our recombination procedure can "safely" merge the antibiotic resistance gene and the gene of interest in one plasmid.


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

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

Functionality of GFP gene from phRGFP plasmid is preserved in the products of recombination with pINS-Puro, pINS-Neo or pINS-Blast plasmids. HeLa cells transfected by 1 mkg of either Target vector phrGFP or the products of recombination (Blast × GFP, Neo × GFP and Puro × GFP). Cells transfected by the recombination products were selected by either blasticidin S, G418 or puromycin and stained by DAPI. Expression of the GFP was analyzed under the microscope. In case of transient transfection by phrGFP vector we usually observed 25% GFP positive cells. In contrast we observed that 80–100% of the cells transfected by the products of recombination and selected by the corresponding antibiotics are GFP positive. Scale bar: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Functionality of GFP gene from phRGFP plasmid is preserved in the products of recombination with pINS-Puro, pINS-Neo or pINS-Blast plasmids. HeLa cells transfected by 1 mkg of either Target vector phrGFP or the products of recombination (Blast × GFP, Neo × GFP and Puro × GFP). Cells transfected by the recombination products were selected by either blasticidin S, G418 or puromycin and stained by DAPI. Expression of the GFP was analyzed under the microscope. In case of transient transfection by phrGFP vector we usually observed 25% GFP positive cells. In contrast we observed that 80–100% of the cells transfected by the products of recombination and selected by the corresponding antibiotics are GFP positive. Scale bar: 20 μm.
Mentions: Then HeLa cells resistant to the antibiotics were inspected under the microscope for the expression of GFP. Only cells transfected by the recombination products were GFP-positive. Moreover, the proportion of the GFP-positive cells was considerably higher than in the case of transient transfection by the phrGFP plasmid (Fig 6). We conclude that our recombination procedure can "safely" merge the antibiotic resistance gene and the gene of interest in one plasmid.

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