Parallel Subcloning of a Test Gene into Multiple DestinationVectors

As an initial demonstration of RC, 12 diverse cloning vectors were converted to Destination Vectors by insertion of a blunt-end cassette comprising attR1-chloramphenicol resistance gene (Cm^R)-ccdB-attR2 (Table ​(Table1).1). Potentially any vector can be similarly converted. To propagate vectors that contain the ccdB gene, we isolated an E. coli strain (DB3.1) containing a gyrA462 mutation that provides resistance to the effects of ccdB. An Entry Clone (~50 ng) containing the chloramphenicol acetyl transferase (CAT) gene flanked by attL sites was separately mixed with each Destination Vector (~50ng) and LR Clonase (a mixture of Int, IHF, and Xis) and incubated for 30 min. An aliquot (1/10 reaction) was then introduced into E. coli DH5α by transformation with selection for colonies containing Ap^R Expression Clones (Fig. ​(Fig.1B).1B). Each reaction generated thousands of transformants. Negative control reactions that lacked CAT Entry Clone DNA gave 200- to 15,000-fold fewer colonies (except CMVneo), indicating that the ccdB gene was effectively inhibiting transformation by unreacted Destination Vectors and, therefore, that most Ap^R colonies contained the desired Expression Clone. The CMVneo Destination Vector was unstable in E. coli and gave a background of ~10%; recently constructed versions show backgrounds similar to other Destination Vectors. All colonies tested contained a plasmid of expected size (four tested per reaction; 48/48), and all had the predicted restriction pattern (two minipreps per reaction, 24/24; data not shown). These results demonstrate parallel transfer of a test gene into multiple Destination Vectors in an efficient and orientation-specific manner with minimal elapsed and hands-on time.

RC-Mediated Cloning of PCRProducts

PCR products flanked by attB sites can be generated by incorporating attB sites (25 base+4 G residues) at the 5′ end of PCR primers: attB1 in the forward primer and attB2 in the reverse primer. These attB-flanked PCR products can be cloned by incubating with attP-containing vectors in the presence of Int and IHF (BP Clonase) to generate Entry Clones (attL1-PCR product-attL2; Fig. ​Fig.1C,D).1C,D). Such clones retain the orientation and reading frame specified by their starting attB sites.

Five human genes (Table ​(Table2)2) were amplified from first-strand cDNA synthesized using total HeLa RNA as a template (Fig. ​(Fig.2A).2A). In addition, the E. coli gus (β-glucuronidase) and tetracycline resistance (tetR) genes were amplified from plasmid clones. All primers contained attB sites. The tetR amplicon contained the natural promoter, ribosome binding site, and stop codon, and thus conferred tetracycline resistance. Amplification of the other six genes began at their methionine start codons and extended to the carboxy-terminal stop codons. Both prokaryotic and eukaryotic translation sequences (Methods) were added between the attB1 site and ATG of the forward PCR primers (except tetR) to allow expression as native protein or amino-terminal fusions in E. coli or eukaryotic cells following transfer from an Entry Clone into the appropriate Destination Vector.

Table 2

Sources ofGenes

Gene	Accession No.	Open Reading Frame size	Protein size
Hu eIF4e	M15353	0.65kb	25kD
Hu tyrosine kinase	U02680	1.1kb	39kD
Hu transferrin receptor	X01060	2.3kb	84kD
Hu β-adaptin	M34175	2.3kb	105kD
Hu MAP4	M64571	3.3kb	121kD
E. coli gus	P05804	1.8kb	68kD
E. coli tetR	J0I749	1.4kb	NA

Open in a separate window

Open in a separate window

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.

Equal volumes (2 μL) of each PCR reaction (40–1000 ng product), 300 ng of attP vector (pDONR203, Fig. ​Fig.2B),2B), and BP Clonase were mixed and incubated for 1 hr, after which an aliquot (1/10 reaction) was introduced into E. coli DH5α cells by transformation, with selection for Km^R (Fig. ​(Fig.2C).2C). The map of one representative clone, pENTR203-eIF4e, is shown in Figure ​Figure2D.2D. Transformants resulting from RC-mediated cloning of the tetR PCR product were found to be tetracycline resistant at high efficiency (195 of 197). Similarly, four colonies from each of the other six cloning reactions (Fig. ​(Fig.2E)2E) showed all 24 of the plasmids with the expected sizes. This approach therefore provides high efficiency cloning of numerous PCR products in parallel with only a few hours of laboratory time. The resulting plasmids contain the amplified DNA in an Entry Clone capable of transferring the insert into any number of Destination Vectors. (We have also constructed vectors that contain multiple cloning sites flanked by attL1 and attL2 sites, allowing Entry Clones to be constructed by standard restriction enzyme cloning.)

METHODS

All materials, including PCR primers, were obtained from Life Technologies, Inc., unless otherwise noted, and were used according to the manufacturers' instructions. RC-related materials are available as the Gateway Cloning Technology. Sequences of DNAs are available at www.lifetech.com. The bacteriophage λ recombination sites used here can be derived from the sequences of the attB1 and attB2 sites (Fig. ​(Fig.1D)1D) and the sequence of the bacteriophage (GenBank accession no. J02459) according to the mechanism of λ recombination (Weisberg and Landy 1983). The attP sites in pDONR203 correspond to λ coordinates 27586 through 27818. The attR sites in the Destination Vectors lack the bases between 27586 and 27618 (deletion of the P1 and H1 domains improves the excision reaction [Bushman et al. 1985]), and base 27630 has been changed to a G to remove an NdeI site. Miniprep DNAs were prepared by alkaline lysis of overnight cultures and dissolved in TE (10 mM Tris HCl pH 7.5, 1 mM EDTA) containing RNase A (Sigma; 1 μg per mL cell culture). Midi and maxi scale DNA preparations used the Concert purification system. Destination Vectors for use in RC may be constructed by inserting the appropriate blunt reading frame cassette (of the general form, attR1−Cm^R−ccdB gene−attR2) into an available restriction site of any plasmid and propagated in E. coli strain DB3.1 (RR1 endA recA gyrA462), which is resistant to the toxic ccdB gene product (Bernard and Couturier 1992).

PCR

Sources of templates are shown in Table ​Table2.2. Primer sequences were, for human eIF4e, [A]atggcgactgtcgaaccggaaa (fwd) and [B]actaaacaacaaacctatttttagtggtgga (rev); for human tyrosine kinase, [A]atgtcccaccagaccggcatc (fwd) and [B]tcagtag tagcttcagtttccgct (rev); for human transferrin receptor, [A]at gatggatcaagctagatcagca (fwd) and [B]actaaaactcattgtcaatgtccc aaacg (rev); for human β-adaptin, [A]atgactgactcaaaatatttcac cac (fwd) and [B]actagttcttgaggatggtctcgtagg (rev); for human MAP4, [A]atggctgacctcagtcttgcag (fwd) and [B]actagatgctt gtctcctg-gatctggc (rev); for E. coli gus, [A]atggtccgtcctgtagaaacc (fwd) and [B]actattgtttgcctccct-gctgcgg (rev); and for E. coli tetR, [A]aattctcatgtttgacagcttatc (fwd) and [B]cgatggatatgttct gccaag (rev). [A]=ggggacaagtttgtacaaaaaagcaggctcattta-actttaagaaggagatatatacc, in which sequences in bold type correspond to attB1 and those following or in italics are translation signals for E. coli or mammalian cells, respectively. [B]=ggggaccactttgtacaagaaagctgggt, in which sequences in bold types correspond to attB2. Primers were desalted but otherwise unpurified. First-strand cDNA (made with ThermoScript reverse transcriptase) equivalent to 200 ng of total HeLa RNA was used as template in the amplification of the human genes; 1 ng of the appropriate E. coli plasmid DNA was used for amplification of gus and tetR genes. Following amplification with Pfx polymerase, amplification products were precipitated with polyethylene glycol+Mg to remove primer dimers (which clone efficiently) and dissolved in TE. Aliquots (2.5 μL) of each product were applied to a 1% agarose/ethidium bromide gel (Fig. ​(Fig.22A).

Clonases

BP Clonase, containing Int and IHF, catalyzes the integrative (BP) reaction (attB×attP→attL+attR). LR Clonase, containing Xis, Int, and IHF, catalyzes the excisive (LR) reaction (attL×attR→attB+attP). IHF (heterodimer; GenBank accession no. X04864 and V00291), Int, and Xis, (bacteriophage λ; GenBank accession no. J02459) were purified from E. coli strains containing the cloned, overexpressed genes. Clonases and other RC materials are available from Life Technologies, Inc., as part of the Gateway Cloning System.

BP Reactions for Cloning PCRProducts

The attP plasmid pDONR203 (300 ng; Fig. ​Fig.2B)2B) was mixed with 2 μL of each purified PCR product in reactions (20 μL) that contained 4 μL BP Clonase in 25 mM Tris HCl pH 7.5, 22 mM NaCl, 5 mM EDTA, 5 mM spermidine HCl, 1 mg/mL BSA. After incubation for 60 min at 25°C, proteinase K (4 μg in 2 μL) was added, and each reaction was incubated at 37°C for 20 min. Aliquots (2 μL) of each reaction were transformed into E. coli DH5α (Library Efficiency) and plated on kanamycin plates (100 μg/mL) Fig. ​Fig.2C)2C) incubated at 37°C. Miniprep DNA was prepared from four colonies from each reaction, and 2μL aliquots were applied to a 1% agarose/ethidium bromide gel (Entry Clones; Fig. ​Fig.22E).

LR (Subcloning)Reactions

The LR reactions described in Table ​Table11 used earlier versions of DNAs, proteins, and reaction conditions. Only the conditions used for the seven genes amplified in Figure ​Figure22 will be described. One miniprep of each cloned gene (Fig. ​(Fig.2E,2E, lanes 1,5,9,13,17,21), and tetR) was chosen as the Entry Clone. Aliquots containing ~200 ng of each miniprep DNA (except ~60 ng and 40 ng of the transferrin receptor and tetR Entry Clones, respectively) were incubated with 300–400 ng of the appropriate Destination Vector (linearized within the chloramphenicol resistance–ccdB region; Fig. ​Fig.3A)3A) in 20 μL reactions containing 4 μL of LR Clonase, 50 mM of Tris HCl pH 7.5, 50 mM of NaCl, 0.25 mM of EDTA, 2.5 mM of spermidine HCl, and 0.2 mg/mL of BSA. Then proteinase K (4 μg in 2 μL) was added, and reactions were incubated at 37°C for 20 min. Aliquots (2 μL) of each reaction were transformed into E. coli DH5α and plated on ampicillin (100 μg/mL) plates (Fig. ​(Fig.2C)2C) incubated at 37°C. Miniprep DNAs of the resulting Expression Clones were prepared from one colony from each reaction, and aliquots were applied to a 1% agarose/ethidium bromide gel (Fig. ​(Fig.33D).

We thank Dr. Howard Nash for generously providing purified λ recombination proteins in early stages of this work and to our colleagues at Life Technologies, Inc., for their many essential contributions.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.