Assisted large fragment insertion by Red/ET-recombination (ALFIRE)―an alternative and enhanced method for large fragment recombineering

Figure 1. Verification of correct pSC101BADγβαA[hygro] (Red/ET (hygro) and pSC101BADγβαA-I-SceI[amp] recombinants. (a ) Correct pSC101BADγβαA[hygro] recombinants were identified by digestion (more ...)

The pSC101BADγβαA-I-SceI[amp] vector (Supplementary Figure 1) was constructed to promote in vivo enzyme digestion followed by recombination and thereby facilitating modification of BAC clones using ALFIRE. It was assembled by introducing the tetracycline (cTc)-inducible I-SceI homing endonuclease fragment from the pST98AS plasmid (16) to the pSC101BADγβαA[hygro] plasmid with standard Red/ET protocols (8). The entire hygro gene was thus exchanged with the I-SceI-Tet-Rep/Tet-Prom-amp unit generating pSC101BADγβαA-I-SceI[amp]. Positive (Ap selected) clones were verified by SspI restriction enzyme digestion (Figure 1 b) and three clones were sequenced over the modified areas. The vector data were submitted to EMBL (AM403094 ).

The vector pSC101BADγβαA-I-SceI[amp] encodes the Redγ, Redβ, Redα and RecA proteins as well as the homing endonuclease I-SceI. Upon activation with L -arabinose and chloro-tetracycline (cTc), respectively, these enzymes will be expressed and cells harbouring this plasmid will become capable of performing digestion and recombination of the resulting ends containing the homologous sequences. Plasmids or BACs containing the 18 bp recognition site of the homing endonuclease I-SceI can be linearized in vivo and the double-strand breaks thus formed promote recombination to identical homology sequences. Efficient recombination is especially important when large DNA fragments are being recombined such as in BAC fusion experiments.

To test the functionality of the I-SceI meganuclease, 10 bacterial colonies harbouring the I-SceI-CS-hmLhcgr -BAC and the pSC101BADγβαA-I-SceI[amp] were grown overnight at 30°C in LB media with Km/Cm/Ap. Next day, three parallel tubes with 1 ml LB media were set up and inoculated with 30 μl of each culture and incubated at 1100 rpm with the following conditions (1) cTc/Cm/Ap, 37°C for 1 h; (2) cTc/Cm/Ap, 30°C for 5 h; (3) Cm/Ap, 30°C for 6 h. None of the ten clones propagated in the cTc-induced tubes incubated at 37°C, showing that I-SceI was expressed and that the I-SceI-CS-hmLhcgr -BAC was linearized at the I-SceI sites. All of the ten clones grew in the non-induced tubes incubated at 30°C indicating that the I-SceI endonuclease was not expressed and that the I-SceI-CS-hmLhcgr -BAC clone and the pSC101BADγβαA-I-SceI[amp] vector could continue to propagate. Another control experiment was also set up where a clone harbouring only the I-SceI-CS-hmLhcgr -BAC was incubated at 37°C in LB media (cTc/Cm) for 6 h, 1100 rpm. This clone also propagated indicating that cTc is not toxic to E. coli at the appointed concentration.

Figure 2. Outline of the ALFIRE procedure. The accepting vector is modified with a counter-selection/selection cassette (RpsL -neo) flanked by two unique restriction sites and containing HAs to the fragment to be inserted. The resulting vector is linearized and (more ...)

In the initial step, a CS cassette flanked by recognition sites of I-SceI and HAs to the insert and the acceptor vector was designed (see Figure 2 and Supplementary Figure 2). The primers were ~140 nt long and this was the limit set by the supplier to guarantee good quality primers. The I-SceI sequence was chosen because the 18 bp long recognition site is rare even in genomic DNA. In fact, the E. coli chromosome does not contain a single I-SceI site and the meganuclease has been used to generate DSBs into foreign DNA amplified in E. coli (16, 17). First, the CS flanked by two I-SceI restriction sites (I-SceI-CS) was inserted to the mouse Lhcgr (mLhcgr) -BAC by Red/ET recombination with the following procedure: the I-SceI-CS cassette was amplified by PCR with primers [CS (I-SceI) to hmLhcgr-F/CS (I-SceI) to hmLhcgr-R] containing (a) the HAs for insertion (~50 nt) to the mLhcgr promoter and 3′ regions; (b) HAs (~55 nt) to subclone the human Lhcgr (hLhcgr ); (c) the 18 nt recognition site of the homing endonuclease I-SceI (TAGGGATAACAGGGTAAT); and (d) the primer sites (24 nt) for the RpsL -neo (Figure 2 and Supplementary Figure 2). Red/ET competent bacteria carrying the mLhcgr -BAC were transformed with the I-SceI-CS PCR product and plated on LB agar plates supplemented with Km/Cm. Positive clones were screened by PCR (primers mLhcgrP-F/mLhcgr3-R) and further selected by their inability to grow (negative selection) on LB agar plates supplemented with Stp/Cm. In addition, the resulting PCR product was digested in vitro with I-SceI (NEB) in order to check for functionality of the inserted I-SceI restriction sites. One of three clones contained a mismatch in the I-SceI sequence leading to reduced or abolished cleavage by this meganuclease. Five positive clones were further selected for sequencing of the entire HA area, which needed to be faultless to allow efficient Red/ET recombination. Four out of five clones were without mismatches and one was selected for further experiments. The I-SceI-CS-hmLhcgr -BAC construct can thus be used as a general PCR template (I-SceI-CS) and the I-SceI recognition sites would not need to be added each time in the oligonucleotide sequences in future experiments, making the oligos shorter and eliminating the need of checking the I-SceI sites every time.

In vitro generation of DSBs

ALFIRE in E. coli

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  • Abstract
  • INTRODUCTION
  • MATERIALS AND METHODS
  • RESULTS
  • DISCUSSION
  • AUTHOR''S CONTRIBUTIONS
  • SUPPLEMENTARY DATA
  • Supplementary Material
  • REFERENCES

DISCUSSION

The use of homologous recombination to generate and manipulate large fragments of DNA has opened up new avenues in the study of functional genomics. A limitation of traditional recombinogenic engineering is the difficulty of inserting large fragments into large episomes in a single step. It is necessary to overcome these limitations in order to facilitate the manipulation of large genomic sequences needed for the production of transgenic, knockout and knock-in organisms.

We have overcome the major difficulty of inserting HAs into large fragments by incorporating the homologous sequences in the acceptor vector together with unique restriction sites thereby promoting generation of DSBs and facilitating recombination. It is important to note, that often such restriction sites are palindromes and therefore the choice of the endonuclease should be carefully addressed to make sure that the resulting ends have little homology in order to reduce vector re-circularization. It is known that as little as ≥6 nt homology near each end might result in ‘end joining’ (8). Homing endonucleases have long recognition sites that are not palindromes and the presence of these sequences is rare even in genomic size DNA. We chose the homing meganuclease I-SceI recognition sequences as unique sites to generate DSBs in the acceptor BAC carrying the HAs for the insert. However, when the entire hLhcgr (~55 kb) was subcloned into the region between the promoter and the polyA of a BAC containing the mouse homologue (mLhcgr ), the efficiency after in vitro digestion (I-SceI) and isolation was relatively low (6.3%). The poorer recombination efficiency is most probably due to the limitations of transforming the large BACs, in particular if they are linear, into bacteria and the risk of shredding of the BAC during the DNA purification steps. To overcome these problems we constructed an all-in-one universal vector (pSC101BADγβαA-I-SceI [amp]) to be used in E. coli. This system expresses the recombinases Redγ, Redβ and Redα as well as the I-SceI homing endonuclease under the tight control of L -arabinose and chloro-tetracycline, respectively, and makes it possible to linearize the acceptor vector intracellularly followed by recombination between homologous sequences. In this way only the hLhcgr BAC was linear resulting in improved transformation efficiency and recombination (69% positive clones). Thus, a 10-fold higher efficiency was achieved by in vivo compared to in vitro DSBs generation by I-SceI (Table 1 ) and the background was also reduced significantly with in vivo enzyme digestion.

We show that by using appropriate unique restriction enzymes sites, and subcloning-HAs, large fragments can be copied in 2 or 3 steps (see Figure 2 and Supplementary Figure 2 for general overview of the ALFIRE method). By adding the HAs for subcloning into the oligos used for amplification of the I-SceI-CS cassette we eliminated the time-consuming use of transfer constructs. The presence of these short exact sequences (~55 nt) is thus sufficient to result in a highly efficient and accurate recombination. We also show that long oligos, up to ~140 nt, can generate satisfactory HAs for insertion and for subcloning. Alternatively, the double HAs could be generated by PCR using four shorter oligos (>80 nt) instead of two in the same PCR reaction.

The methodology described is highly efficient and an easy protocol to follow in order to insert, fuse, exchange and manipulate virtually any size of DNA or plasmid without long template PCR and undesired selection markers. It is thereby an extra tool in addition to existing methods (8,16,