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. 2000 Nov;10(11):1788-95.
doi: 10.1101/gr.143000.

DNA cloning using in vitro site-specific recombination

J L Hartley  1 , G F Temple, M A Brasch
Affiliations

DNA cloning using in vitro site-specific recombination

J L Hartley et al. Genome Res. 2000 Nov.

Abstract

As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function. Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis. We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. Numerous DNA segments can be transferred in parallel into many different vector backgrounds, providing an approach to high-throughput, in-depth functional analysis of genes and rapid optimization of protein expression. The resulting subclones maintain orientation and reading frame register, allowing amino- and carboxy-terminal translation fusions to be generated. In this paper, we outline the concepts of this approach and provide several examples that highlight some of its potential.

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Figures

Figure 1
Figure 1
(A) Schematic of gene cloning and transfer by recombinational cloning. The triangles represent recombination sites. Genes are cloned into Entry Vectors by in vitro recombination of PCR products or with restriction enzymes and ligase. Thereafter, genes are moved entirely by recombination. (B) Subcloning of a gene from the Entry Vector into a Destination Vector. (C) Cloning attB-PCR products by in vitro recombination. (D) Sequences of the attB1 and attB2 sites that flank the gene either in a PCR product made with primers containing attB sites or in an Expression Clone.
Figure 2
Figure 2
(A) Agarose gel (1%) of PCR amplification products (2.5 μL of 35 μL) using attB-primers (see Methods) encoding eIF4e (lane 1), tyrosine kinase (lane 2), transferrin receptor (lane 3), β-adaptin (lane 4), MAP4 (lane 5), glucuronidase (Gus, lane 6), or the tetracycline-resistance gene (TetR; lane 7). (B) attP cloning vector pDONR203. PCR products cloned by in vitro recombination replace the chloramphenicol-resistance and ccdB genes; the recombination reactions convert the attP sites to attL sites. (C) Colonies resulting from transformation of E. coli with reactions (2 μL of 22 μL) containing the PCR products, pDONR203, and BP Clonase. The negative control contained all components except PCR product. (D) Entry Clone pENTR203-eIF4e, the product of recombinational cloning of the eIF4e PCR product into pDONR203. (E) Miniprep DNA (Entry Clones) from the colonies in C. Lanes 1–4, eIF4e; lanes 5–8, tyrosine kinase; lanes 9–12, transferrin receptor; lanes 13–16, β-adaptin; lanes 17–20, MAP4; lanes 21–24, Gus; M, supercoiled DNA ladder.
Figure 3
Figure 3
(A) Destination Vector pDEST17, for expressing His6 fusions in Escherichia coli. (B) Colonies resulting from transformation of DH5α cells with 2 μL (of 22 μL) of LR reactions. The negative control contained all components except the Entry Clone DNA. pDEST17 was linearized at the unique NcoI site prior to mixing with Entry Clone DNA. (C) The Expression (attB) clone pEXP17-eIF4e, which resulted from the LR reaction with pENTR203-eIF4e and pDEST17. The subcloned eIF4e gene has replaced the chloramphenicol resistance-ccdB segment, with the amino end of the gene downstream of the T7 promoter and the His6 tag in frame with the eIF4e open reading frame. The recombination reactions convert the attR sites to attB sites (25 bp). (D) Miniprep DNA of single colonies (Expression Clones) from each reaction. Lane 1, eIF4e; lane 2, tyrosine kinase; lane 3, transferrin receptor; lane 4, β-adaptin; lane 5, MAP4; lane 6, Gus; lane 7; TetR. M, supercoiled DNA ladder.
Figure 4
Figure 4
(A) SDS-PAGE gel of proteins expressed from Expression Clones of the genes in pDEST17 (His6 fusion) vector in Escherichia coli strain BL21 SI. (Lane 1), eIF4e; (lane 2), tyrosine kinase; (lane 3), transferrin receptor; (lane 4), β-adaptin; (lane 5), MAP4 (MAP4 is known to migrate aberrantly on SDS-PAGE gels; Chapin et al. 1995); (lane 6), Gus. (B) SDS-PAGE gel of expression of genes subcloned from the same Entry Clones as in (A) into baculovirus Destination Vectors. (Lanes 1–5) are pDest8 (native expression; (lane 1), eIF4e; (lane 2), tyrosine kinase; (lane 3), transferrin receptor; (lane 4), β-adaptin; (lane 5), MAP4). (Lanes 6–8) the gus gene, from the same Entry Clone as in A, expressed in the baculovirus vectors pDest8 (lane 6, native, 68 kD), pDest10 (lane 7, His6 fusion, 74 kD), and pDest20 (lane 8, GST fusion, 97 kD). As in E. coli expression (above), MAP4 protein migrated as a ∼200-kD protein instead of as a 121-kD protein (Chapin et al. 1995).
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