Download Frequently Asked Questions about Red/ET Cloning

How does the Red/ET Recombination system compare to other
recombination systems, ie, Gateway, Cre-Lox, RecA/χ, RecA?
Gene Bridges’ proprietary Red/ET Recombination technology (e.g.
Angrand et al 1999) has been published under several names:
•
•
•
•
ET cloning/ ET recombination (e.g. Muyrers et al. 2000)
GET recombination (e.g. Nefedov et al. 2000)
Recombineering (e.g. Copeland et al. 2001)
λ Red-mediated recombination (e.g. Murphy and Campellone 2003)
Red/ET Recombination is homologous recombination mediated by RecET
or Redαβ and it utilizes short homology sequences to accomplish the
recombination. Gateway and Cre-Lox systems are based on site-specific
recombination. They have a different underlying recombination
mechanism compared with Red/ET Recombination.
The RecA/χ system is based on homologous recombination mediated by
RecBCD in E.coli. It is dependent on a short sequence called chi (χ)
present in the molecules. The homology sequences needed in RecA/χ
system are much longer than the ones used in Red/ET Recombination.
RecA mediated homologous recombination (Yang et al., Nature
Biotechnology, 1997) is mediated by RecA alone and is dependent on
longer homology sequences. The crucially longer homology sequences
cannot be synthesized as oligonucleotides. Compared with Red/ET
Recombination, it needs more conventional steps to pre-condition the
recombinogenic molecules.
Functionally?
Any E.coli strain can be converted in a Red/ET Recombination proficient
strain. Seamless cloning is achieved, since Red/ET Recombination is not
dependent on standard sequences like restriction sites, loxP, FRT, attsites,…
No site restriction – Red/ET functions at any point, independent of
available restriction sites.
Red/ET can be applied with great ease to directly subclone or modify the
bacterial chromosome resulting in designed bacterial strains.
Red/ET is one system for almost all applications: insertions, deletions from
1 bp up to 250 kb, substitutions, fusions, subcloning, BAC stitching.
Almost no size limitation.
1
Red/ET Recombination technology circumvents preparative DNA steps,
since the DNA stays inside the bacterial cell.
Short sequence motives like restrictions sites, loxP, FRT, IRES can be
introduced together with the actual DNA modification step.
Efficiency of Red/ET Recombination?
Recombination events per cell
2 end-mediated recombination event, due to introduction of
linearized DNA
Absolute efficiency of recombination without selection can reach up to 5%
Recombination events vs background under selective conditions > 95%
Fidelity (are open reading frames maintained when recombination occurs
within coding sequence?
Yes, since Red/ET Recombination is a nucleotide-precise cloning
technology.
Ease of use?
Red/ET Recombination is an established technology based on standard
procedures like generation of competent cells, PCR, DNA mini preparation,
gel electrophoresis.
Limitations and biases vs advantages
Limitations/biases:
Red/ET cloning of repetitive elements, identical cassettes
The need for retransformation in case of high copy plasmids
Advantages:
unknown sequences can be cloned, only the sequence information of the
homology arms is needed
Short sequence motives like restrictions sites, loxP, FRT, IRES can be
introduced together with the actual DNA modification step
2
unlike site specific recombination no standard sequences will be
introduced
What is the size range of sequence that can be cloned?
Cloning 1bp up to 250 kb; Subcloning size dependent on origin of
replication; subcloning of 80 kb has been confirmed.
What is the length of homology required?
30-60 bp is recommended
Is electroporation required for cloning? Can high throughput cloning be
done with chemically competent cells?
For a highly efficient performance electroporation is recommended
resulting in an efficiency of 5x105 colonies per electroporation.
Chemically competent cells can be used for high throughput cloning, an
efficiency of 103 colonies per electroporation can be reached under such
conditions. Additionally, there is a limitation regarding the size of DNA to
be incorporated.
Can the system be used to shuttle sequence between vectors?
Yes, via Red/ET subcloning. The recipient vector needs to exert a different
origin of replication than the donor vector. In pilot studies it was shown
that Red/ET technology can be applied to shuttle between different vector
sets.
Via Red/ET subcloning in two steps (preventing from PCR of recipient
vector)
Has Gene Bridges tested the difference between efficiency of
recombination where recombination-specific plasmids which are situated
on a plasmid versus embedded in the bacterial chromosome?
A bacterial strain expressing Red/ET proteins directly from the
chromosome like YZ2000 shows comparable efficiency to a bacterial
strain electroporated with a Red/ET proficiency plasmid. In the latter case,
the recombination system is only active for a short period of time so that
the integrity of the DNA of interest is maintained. By applying Red/ET
proficiency plasmids one can use any bacterial strains the lab is familiar
with. This is an advantage especially when using bacterial BAC host
strains or chromosomal modifications.
3
Has Gene Bridges isolated the recombination-specific proteins and
demonstrated in vitro application?
Pilot studies have been undertaken, so far without success. Gene Bridges
is studying the co-factors of Red/ET Recombination in E.coli to accomplish
the goal of in vitro application.
Can the recombination-specific proteins function in eukaryotic cells? Can
you provide examples of successful application in eukaryotic cells? How
efficient is recET/Red in eukaryotic cells, if applicable?
Yes, please refer to our publication (BMC Biotechnology, 2003). Gene
Bridges has also more indications though the data hasn´t been published
yet. At this moment, we only can achieve a limited number of exercises in
eukaryotic cells and the efficiency is still low.
In a traditional cloning situation, ie, linearized vector with homologous
ends + insert with complimentary homologous ends, is there a difference in
recombination efficiency when using a gel-isolated fragment and a PCRgenerated fragment?
In theory, there is no impact on Red/ET performance using either an
isolated DNA fragment or a PCR product. But a gel-isolated fragment
exerts several disadvantages, such as EtBr staining, shearing and often a
lower concentration. Normally, it shows a bit lower efficiency than a PCRgenerated fragment.
A sometimes observed, higher efficiency after precipitation, maybe due to
a higher DNA concentration that has been applied, to prevent from
unsatisfying yield.
You once marketed several kits, including kits for standard cloning (and I
believe, mutagenesis). It is noted that you now only market a BAC cloning
kit. Is there a reason for this?
The applications of the kits have become more focused. Each kit includes
a standard control experiment. The application of Red/ET in other fields
can be accomplished with these kits as well (see PDF Document). Gene
Bridges is developing several new kits with defined focuses. New kits are
to be launched in the near future:
•
•
•
Fragment cloning (applicable for isolated and PCR generated DNA)
Targeting vector for conditional KO
Modification of the bacterial chromosome
4
References and Patents
•
Angrand P.O., Daigle N., van der Hoeven F., Scholer H.R., Stewart A.F.
Simplified generation of targeting constructs using ET recombination.
Nucleic Acids Res 27, e16 (1999).
•
Copeland NG, Jenkins NA, Court DL: Recombineering: a powerful new
tool for mouse functional genomics. Nat Rev Genet, 2:769-779 (2001).
•
Murphy K.C. and Campellone K.G. Lambda Red-mediated
recombinogenic engineering of enterohemorrhagic and enteropathogenic
E. coli. BMC Mol Biol. Dec 13;4(1):11. (2003).
•
Muyrers, J.P.P., Zhang, Y., Buchholz, F., Stewart, A.F. RecE/RecT and
Redα/Redβ initiate double-stranded break repair by specifically interacting
with their respective partners. Genes Dev 14, 1971-1982 (2000).
•
Muyrers J.P.P., Zhang Y., Benes V., Testa G., Ansorge W., Stewart A.F.
Point mutation of bacterial artificial chromosomes by ET recombination.
EMBO Reports 1, 239-243 (2000).
•
Muyrers, J.P.P., Zhang, Y., Stewart, A.F. ET cloning: Think recombination
first. Genetic Engineering, Principles and Methods (Ed. J.K. Setlow), 22,
77-98 Kluwer Academic/Plenum Publishers, NY. (2000).
•
Nevedov M; Williamson R, Ioannou P.A.. Insertion of disease-causing
mutations in BACs by homologous recombination in Escherichia coli.
Nucleic Acids Res. Sep 1;28 (17):E79 (2000).
•
Yang X.W., Model P., Heintz N. Homologous recombination based
modification in Escherichia coli and germline transmission in transgenic
mice of a bacterial artificial chromosome. Nature Biotechnol. 15,859-865
(1997).
Patents
•
Stewart, A.F., Zhang, Y., and Buchholz, F. 1998. Novel DNA cloning
method. European Patent No.1034260 (issued on 12th of March, 2003),
United States Patent No 6,509,156.
•
Stewart, A.F., Zhang, Y., and Muyrers, J.P.P. 1999. Methods and
compositions for directed cloning and subcloning using homologous
5
recombination. United States Patent No. 6,355,412 (issued on 12th of
March, 2002).
6