DNA cloning by homologous recombination in Escherichia coli

Zhang, Y.; Muyrers, Joep P P; Testa, Giuseppe; Stewart, A. Francis

doi:10.1038/82449

Cited by 370 publications

(314 citation statements)

References 20 publications

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“…To date, these cloning techniques include the restriction enzyme cloning, 1 blunt-end cloning, 2 TA cloning, 3 classical ligation-independent cloning (LIC), [4][5][6][7][8][9][10][11] hybridization cloning, 12,13 and in vivo cloning. [14][15][16] In fact, the last two methods are also LIC because they are ligase-free cloning techniques. All these methods require the specific enzymatic treatment of target gene and vector, 1,[5][6][7][8][9] the use of primers carrying modified bases, 4,[9][10][11] or some specific bacterial strains, [14][15][16] except for hybridization cloning.…”

Section: Introductionmentioning

confidence: 99%

“…[14][15][16] In fact, the last two methods are also LIC because they are ligase-free cloning techniques. All these methods require the specific enzymatic treatment of target gene and vector, 1,[5][6][7][8][9] the use of primers carrying modified bases, 4,[9][10][11] or some specific bacterial strains, [14][15][16] except for hybridization cloning. Among them, the LIC being easy to carry out can be easily adopted for high-throughout cloning.…”

Section: Introductionmentioning

confidence: 99%

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The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Liu

2010

Protein Science

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Function studies of many proteins are waited to develop after genome sequencing. Highthroughout technology of gene cloning will strongly promote proteins' function studies. Here we describe a ligation-independent cloning (LIC) method, which is based on the amplification of target gene and linear vector by PCR using phosphorothioate-modified primers and the digestion of PCR products by k exonuclease. The phosphorothioate inhibits the digestion and results in the generation of 3 0 overhangs, which are designed to form complementary double-stranded DNA between target gene and linear vector. We compared our phosphorothioate primer cloning methods with several LIC methods, including dU primer cloning, hybridization cloning, T4 DNA polymerase cloning, and in vivo recombination cloning. The cloning efficiency of these LIC methods are as follows: phosphorothioate primer cloning > dU primer cloning > hybridization cloning > T4 DNA polymerase cloning >> in vivo recombination cloning. Our result shows that the 3 0 overhangs is a better cohesive end for LIC than 5 0 overhang and the existence of 5 0 phosphate promotes DNA repair in Escherichia coli, resulting in the improvement of cloning efficiency of LIC. We succeeded in constructing 156 expression plasmids of Aeropyrum pernix genes within a week using our method.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Liu

2010

Protein Science

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“…Another variation that we have begun to use for other humanization projects is the consolidation of the equivalent of steps 1 and 2 of Fig. 3 into a single step by using a "gap repair" fragment similar to methods described by Zhang et al (38). …”

Section: Bac Recombineering To Generate Large Compound Bac-based Targmentioning

confidence: 99%

Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes

Macdonald

Karow

Stevens

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

290

273

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Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized. Heretofore, most genetic humanizations have involved disruption of the endogenous mouse gene with simultaneous introduction of a human transgene at a new and random location (so-called KO-plus-transgenic humanization). More recent efforts have attempted to replace mouse genes with their human counterparts at the same genetic location (in situ humanization), but such efforts involved laborious procedures and were limited in size and precision. We describe a general and efficient method for very large, in situ, and precise genetic humanization using large compound bacterial artificial chromosome-based targeting vectors introduced into mouse ES cells. We applied this method to genetically humanize 3-Mb segments of both the mouse heavy and κ light chain Ig loci, by far the largest genetic humanizations ever described. This paper provides a detailed description of our genetic humanization approach, and the companion paper reports that the humoral immune systems of mice bearing these genetically humanized loci function as efficiently as those of WT mice.genome engineering | therapeutic antibody | immunoglobulin locus T he laboratory mouse is one of the premier model organisms used by biologists. As a mammal, the mouse is more genetically similar to humans and thus more relevant to human physiology and disease than many other model organisms. Its small size, short generation time, and the availability of a large variety of inbred strains have led to a robust body of classical genetic research on the mouse. The utility of mice as a genetic model is also greatly enhanced by powerful transgenic and knockout technologies, allowing researchers to study the effects of the directed overexpression or deletion of specific genes. However, despite all of its advantages, the mouse remains an imperfect model of human disease and an imperfect platform on which to test potential human therapeutics. One issue is that, although about 99% of human genes have a mouse homolog (1), potential therapeutic agents often do not cross-react, or cross-react only poorly, with the mouse ortholog of the intended human target. To overcome this problem, selected target genes can be humanized, that is, the mouse gene can be eliminated and replaced by the corresponding human orthologous gene sequence. Because of the difficulties of using conventional KO technologies to directly replace large mouse genes with their large human genomic counterparts, genetic humanization is currently mo...

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“…This procedure is much more efficient than those previously used to disrupt Streptomyces genes (3). Our approach was based on the discovery that allelic exchanges on the Escherichia coli chromosome can be achieved by recombination with a PCR-generated selectable marker flanked at both ends by extensions of only a few tens of nucleotides homologous to the desired region of the chromosome, when the Red␣ (exo), Red␤ (bet), and Red␥ (gam) proteins of the phage are present in the targeted strain (4)(5)(6)(7)(8)(9). We have used -Red to promote recombination in E. coli between a PCR-amplified antibiotic resistance cassette selectable in E. coli and Streptomyces, and S. coelicolor DNA on a cosmid from the set used to sequence the S. coelicolor genome (10).…”

mentioning

confidence: 99%

PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

Gust

Challis

Fowler

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

1,315

1,554

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Streptomycetes are high G؉C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and -Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriTRK2 for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT RK2 to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.FLP recombinase ͉ lambda Red recombinase ͉ oriT ͉ SCO5222 ͉ SCO6073

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DNA cloning by homologous recombination in Escherichia coli

Cited by 370 publications

References 20 publications

The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes

PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

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