Construction of Gene-Targeting Vectors by Recombineering

Reagents

Agarose gels (1%) and reagents for electrophoresis

Antibiotics

Ampicillin (Amp), 50 µg/mL
 

Chloramphenicol (Cm), 12.5 µg/mL
 

Kanamycin (Kan), 30 µg/mL
 

Hygromycin B (Hygro), 75 µg/mL
 

Concentrations indicated are final concentrations used in both liquid and solid media.

^{BAC cells (containing genomic region of interest)}

^{Bovine serum albumin (BSA), 100X (supplied with restriction enzymes from New England Biolabs [NEB])}

^{DH10B electrocompetent cells (Invitrogen)}

^{Exonuclease I (10U; NEB)}

^{Extensor Hi-Fidelity PCR Master Mix (ABgene)}

^{Fast-Media and agar (InvivoGen)}

^{This is used to prepare media containing one of the following:}

^{Blasticidin S hydrochloride (Blasticidin; Bsd)}

^{Puromycin (Puro)}

^{Prepare agar and TB (liquid medium) according to manufacturer’s instructions on packet. Do not overheat the mixture. Cool the media rapidly in ice slurry for 5 min after heating in a microwave. Rapid cooling of the heated mixture reduces degradation of the antibiotics. We find that Puro selection in E. coli cells is not as stringent as other commonly used antibiotics. Therefore, we do not recommend the use of Puro selection in 96-well liquid media culture.}

^{As an alternative to using the Fast-Media Kit, one can prepare media using 50-100 µg/mL of Bsd or 1-5 µg/mL of Puro.}

^{HPLC (high performance liquid chromatography)-purified primers (Sigma-Genosys) for isolation of selection cassettes (see Step 2)}

^{HPLC-purified long oligos perform better than desalted-only oligos. HPLC presumably eliminates most of the incorrect oligos that are present in significant amounts in the crude desalted oligos.}

^{LB agar}

^{LB agar is used to prepare solid media for this protocol.}

^{Low-salt LB liquid medium}

^Plasmids

^{Recombineering plasmid, pSim18}

^{Retrieval vector, PL611}

^{Selection cassette plasmids, Bsd and Neo cassettes}

^{LacZ reporter cassette plasmid, PL613}

^{Negative selection plasmid, Cm-MC1TK}

^{These plasmids can be obtained from the Sanger Institute Archive Team ( http://www.sanger.ac.uk ). pSim18 is extracted from E. coli cells using a QIAprep Spin Miniprep Kit. Because it is a low-copy plasmid, the yields are usually lower than regular high-copy plasmids such as pBluescript.}

^{QIAprep Spin Miniprep Kit (QIAGEN)}

^{QIAquick Gel Extraction Kit (QIAGEN)}

^{Restriction enzymes and buffers}

^{I-SceI, I-CeuI, BamHI, SalI, EcoRI, NotI (NEB)}

^{I-PpoI (Promega)}

^{Selection plates}

^LB-Amp

^LB-Bsd

^LB-Kan

^LB-Hygro

^LB-Kan-Cm

^LB-Puro-Kan

^{Prepare LB-Amp, LB-Kan, LB-Hygro, and LB-Kan-Cm plates using LB agar and antibiotic concentrations specified above. Prepare LB-Bsd plates using Fast-Media Kit. Prepare LB-Puro-Kan plates using Fast-Media Kit and add Kan to final concentration of 30 µg/mL.}

^{SOC medium (Invitrogen)}

^{TOP10 chemical-competent cells (Invitrogen)}

^{T4 DNA ligase and buffer (NEB)}

^Equipment

^{Electroporation cuvette (0.1-cm gap) (Bio-Rad)}

^{Equipment for agarose gel electrophoresis}

^{Gene Pulser Xcell Electroporation System (Bio-Rad)}

^{Heat block (Grant Instrument)}

^Ice

^{Incubator (microbiological) preset to 37°C}

^{Incubators (shaking) preset to 32°C and 37°C}

^{Microcentrifuge (benchtop) with cooling system}

^{Microcentrifuge tubes (1.5 mL)}

^{Polypropylene tubes (15 mL)}

^{PTC-225 PCR (polymerase chain reaction) machine (MJ Research)}

^METHOD

^{PCR Amplification of the Selection Cassettes (3 h)}

^{1. Set up restriction digestion of the selection cassettes and retrieval vector (1.0 µg) with 1 µL of each enzyme, 3 µL of the appropriate NEB buffer, 0.3 µL of BSA, and water to 30 µL total volume. Incubate reactions at 37°C for 2 h.}

Cassette Enzymes to use Bsd cassette EcoRI and BamHI Neo cassette NotI and SalI PL611 EcoRI and BamHI

 

^{2. Run the digestion products through a 1% agarose gel and excise bands corresponding to 560 bp (Bsd ), 2 kb (Neo ), and 3 kb (PL611). Purify them using a QIAquick Gel Extraction Kit.
To avoid background from uncut cassettes of the PCR templates, transform 1.0 ng of the restriction-digested and gel-purified retrieval vector and the two selection cassettes into DH10B cells. If there are any background drug-resistant colonies, repurify the cassettes and test again until clean.}

^{3. Set up PCR amplification of the selection cassettes and retrieval vector:}

^{i. Use the following reagents in each reaction mixture:
1.0 ng of digested template
25 µL of the Extensor Hi-Fidelity PCR Master Mix
2 µL of each primer (10 µM)
20 µL of PCR-grade water}

^{ii. Use the following PCR conditions:
One cycle of 94°C for 4 min
35 cycles of 94°C for 30 sec, 60°C for 30 sec, and 68°C for 1 min (Bsd ) or 2-3 min (Neo and retrieval backbone)
One cycle of 68°C for 5 min}

^{4. After PCR reactions, treat as follows:}

^{i. Add 0.5 µL of Exonuclease I per 50 µL of PCR products.}

^{ii. Incubate at 37°C for 1 h.}

^{iii. Heat inactivate at 80°C for 20 min.}

^{5. Run 1 µL of PCR reaction in a 1% agarose gel to check for PCR products. Purify the rest of the PCR reactions using a QIAprep Spin Miniprep Kit, and elute in 50 µL of PCR-grade water.}

^{Conferring Recombineering Competence to BAC-Harboring Cells (2 d)}

^{6. Inoculate BAC cells (containing genomic region of interest) into 1.0 mL of low-salt LB liquid medium with Cm (in a 15-mL polypropylene tube). Grow cells overnight at 37°C with shaking at 200 rpm.}

^{7. Transfer cells to a 1.5-mL microcentrifuge tube and collect cells by centrifuging the tube at maximum speed using a benchtop centrifuge for 25 sec.}

^{8. Decant supernatant and wash three times with ice-cold water. Pellet cells by centrifuging at maximum speed using a benchtop centrifuge for 25 sec at each wash step.}

^{9. Resuspend cells in 50 µL of ice-cold water with 1 ng of pSim18 and electroporate using the Gene Pulser Xcell Electroporation System (1.75 kV, 25 µF with the pulse controller set at 200 ).}

^{10. Add 1.0 mL of low-salt LB liquid medium to the cuvette and incubate the transformation mixture at 32°C for 1 h.
In this and subsequent steps where no antibiotic is specified, use low-salt LB liquid medium with no antibiotic.}

^{11. Plate out cells onto an LB-Hygro plate and incubate the plate at 32°C overnight.}

^{Targeting Selection Cassettes to BACs (2 d)}

^{12. Pick one HygroR BAC colony, inoculate into 1.0 mL of LB with Cm and Hygro (in a 15-mL polypropylene tube), and incubate overnight at 32°C with shaking at 200 rpm.}

^{13. Inoculate 25, 35, 45, and 55 µL of overnight culture into four 15-mL polypropylene tubes, each containing 1.0 mL of fresh LB, and incubate at 32°C with shaking at 200 rpm for 2 h.}

^{14. Transfer all four cultures to separate wells of a 42°C heat block and incubate for 15 min.}

^{15. Place the heat block directly onto ice and incubate for 5 min.}

^{16. Transfer cells into four 1.5-mL microcentrifuge tubes and centrifuge at maximum speed using a benchtop centrifuge for 25 sec.}

^{17. Decant supernatant and wash three times with ice-cold water. Pellet cells by centrifuging at maximum speed using a benchtop centrifuge for 25 sec at each wash step.}

^{18. Combine cells from all four tubes, resuspend them in 50 µL of ice-cold water with ~300 ng-1.0 µg of PCR product (Bsd cassette), and perform electroporation.}

^{19. Add 1.0 mL of LB to the cuvette and incubate the transformation mixture at 32°C for 1 h.}

^{20. Plate out the cells onto an LB-Bsd plate and incubate the plate at 32°C overnight.}

^{21. Pick 10 BsdR colonies and streak these colonies onto two separate plates, LB-Amp and LB-Hygro. Incubate the plates at 32°C overnight.
The desired colonies should be AmpS and HygroR . Sensitivity to Amp indicates that these BsdR colonies are true targeted clones and not contamination from the original Bsd plasmid. AmpS -HygroR colonies still retain pSim18 and can be used directly for the next round of recombineering.
See Troubleshooting.}

^{22. Use the BsdR -AmpS -HygroR colonies for the next round of recombineering to target the Neo cassette to the BAC (repeat Steps 7-16).}

^{23. The BsdR -KanR -AmpS -HygroR clones are now ready for retrieval (Step 24).}

^{Retrieving Genomic DNA to a Plasmid Backbone (2 d)}

^{24. Inoculate one BsdR -KanR -AmpS -HygroR colony into 1.0 mL of LB with Kan in a 15-mL polypropylene tube and incubate the tube overnight at 32°C with shaking at 200 rpm.}

^{25. Inoculate 25, 35, 45, and 55 µL of overnight culture into four 15-mL polypropylene tubes, each containing 1.0 mL of fresh LB, and incubate at 32°C with shaking at 200 rpm for 2 h.}

^{26. Transfer all four cultures to separate wells of a 42°C heat block and incubate for 15 min.}

^{27. Place the heat block directly onto ice and incubate for 5 min.}

^{28. Transfer cells into four 1.5-mL microcentrifuge tubes and centrifuge at maximum speed using a benchtop centrifuge for 25 sec.}

^{29. Decant supernatant and wash three times with ice-cold water. Pellet cells by centrifuging at maximum speed using a benchtop centrifuge for 25 sec at each wash step.}

^{30. Combine cells from all four tubes, resuspend in 50 µL of ice-cold water with ~300 ng-1.0 µg of PCR product (PL611 retrieval cassette), and perform electroporation.}

^{31. Add 1.0 mL of LB to the cuvette and incubate the transformation mixture at 32°C for 1 h.}

^{32. Plate out cells onto an LB-Amp plate and incubate the plate at 32°C overnight. There are usually hundreds of AmpR colonies on the plate after 12-16 h of incubation.
In some cases, most colonies arise due to either self-ligation or intramolecular recombination of the retrieval plasmid vector. However, the true retrieval plasmid containing the required product can be easily identified from the background AmpR colonies using an additional retransformation because only the correct retrieved plasmid carries the Bsd and Neo cassettes.
See Troubleshooting.}

^{33. Add 2 mL of LB to the plate and swirl the plate to collect the cells.}

^{34. Isolate the plasmids from the cell mixture using a QIAprep Spin Miniprep Kit. Use 1.0 µL of the plasmid preparation for transformation into DH10B electrocompetent cells.}

^{35. Add 1.0 mL of LB to the cuvette and incubate the transformation mixture at 37°C for 1 h.}

^{36. Plate out cells onto an LB-Kan plate and incubate the plate at 37°C overnight.}

^{37. Inoculate three KanR colonies into 3 mL of LB with Kan in a 15-mL polypropylene tube and incubate the tubes overnight at 37°C with shaking at 200 rpm.}

^{38. Isolate the plasmids from the cell mixture using a QIAprep Spin Miniprep Kit and check these plasmids using restriction digestion with appropriate enzymes. These KanR colonies are the correctly retrieved plasmids.}

^{Replacement of Bsd Cassette with the lacZ Reporter (2 d)}

^{39. Set up restriction reactions of the retrieved plasmid (1.5 µg) or PL613 (1.5 µg) with the following reagents:}

^{2 µL of I-SceI
1 µL of I-CeuI
3 µL of NEB buffer 4
0.3 µL of BSA
Water to 30 µL total volume
Incubate reaction at 37°C for 2 h.
The activity of I-SceI in NEB buffer 4 is 50%; therefore, we use twice as much I-SceI in the double digestion reaction with I-CeuI.}

^{40. Purify the restriction-digested retrieved plasmid using a QIAprep Spin Miniprep Kit and elute DNA in 30 µL of PCR-grade water.}

^{41. Run the restriction digestion reaction of PL613 through a 1.0% agarose gel, and purify the lacZ reporter cassette (7 kb band) from the backbone using a QIAquick Gel Extraction Kit.}

^{42. Set up the ligation reaction of purified digestion products as follows:}

^{12 µL of lacZ reporter cassette (600 ng)
10 µL of purified retrieved plasmid (50 ng)
2.5 µL of T4 DNA ligase buffer
1.0 µL of T4 DNA ligase
Incubate the reaction at room temperature (25°C) for 2 h.
We find that increasing the molar ratio of the insert ( lacZ reporter cassette) to the vector (retrieved plasmid backbone) to 10:1 results in a dramatic increase in the number of PuroR -KanR colonies. This is probably due to the competition of the lacZ reporter fragment (7 kb) with the Bsd fragment (0.6 kb) for ligation with the retrieved plasmids.}

^{43. Add 5 µL of the ligation products to chemical-competent TOP10 cells and incubate on ice for 30 min.}

^{44. Heat-shock the cells at 42°C without shaking for 30 sec.}

^{45. Add 250 µL of SOC and incubate the transformation mixture at 37°C for 1 h.}

^{46. Plate out cells onto an LB Puro-Kan plate and incubate the plate at 37°C overnight.
Colonies are typically observed after 16-24 h.
See Troubleshooting.}

^{47. Inoculate four PuroR -KanR colonies into 3 mL of LB (in 15-mL polypropylene tubes) with Kan, and culture at 37°C overnight with shaking at 200 rpm.}

^{48. Isolate the plasmids using a QIAprep Spin Miniprep Kit and set up restriction digestion with I-SceI and I-CeuI to confirm the identity of the plasmids.}

^{Targeting the Negative Selection Cassette to the Vector Plasmid Backbone (2 d)}

^{49. Inoculate 25, 35, 45, and 55 µL of overnight culture into four 15-mL polypropylene tubes, each containing 1.0 mL of fresh LB, and incubate at 32°C with shaking at 200 rpm for 2 h.}

^{50. Transfer all four cultures to separate wells of a 42°C heat block and incubate for 15 min.}

^{51. Place the heat block directly onto ice and incubate for 5 min.}

^{52. Transfer cells into four 1.5-mL microcentrifuge tubes and centrifuge at maximum speed using a benchtop centrifuge for 25 sec.}

^{53. Decant supernatant and wash three times with ice-cold water. Pellet cells by centrifuging at maximum speed using a benchtop centrifuge for 25 sec at each wash step.}

^{54. Combine cells from all four tubes, resuspend in 50 µL of ice-cold water with ~10-100 ng of purified Cm-MC1TK cassette, and perform electroporation.}

^{55. Add 1.0 mL of LB to the cuvette and incubate the transformation mixture at 32°C for 1 h.}

^{56. Plate out cells onto an LB-Kan-Cm plate and incubate the plate at 32°C overnight.}

^{57. Inoculate several PuroR -KanR -CmR colonies into 3 mL of LB with Kan in 15-mL polypropylene tubes and grow at 37°C overnight with shaking at 200 rpm.
See Troubleshooting.}

^{58. Isolate plasmid DNA using a QIAprep Spin Miniprep Kit and dilute the plasmid DNA 1:100.}

^{59. Add 1.0 µL of the diluted DNA to electrocompetent DH10B cells and perform electroporation.}

^{60. Add 1.0 mL of LB to the cuvette and incubate the transformation mixture at 37°C for 1 h.}

^{61. Plate out the cells onto an LB-Kan plate.
This retransformation step eliminates plasmids multimers formed during recombineering.}

^{62. Inoculate KanR colonies into 3 mL of LB with Kan in 15-mL polypropylene tubes and grow at 37°C overnight with shaking at 200 rpm.}

^{63. Isolate plasmid DNA using a QIAprep Spin Miniprep Kit to obtain the final targeting vector.}

^{Verification of the Final Targeting Construct (3 h)}

^{64. Set up restriction digestions using 1.0 µL of the final targeting vectors with either 2 µL of I-PpoI or a combination of 2 µL of I-SceI and 1 µL of I-CeuI in a total reaction volume of 30 µL. Incubate the reaction mixture at 37°C for 2 h.}

^{65. Run the restriction digestion products through a 1% agarose gel to check for expected restriction digestion patterns.}

^{66. Sequence the final targeting vectors to verify the key DNA junctions, including the two junctions between the plasmid backbone and the two ends of the retrieved genomic DNA fragment, and the lox P, FRT, and F3 sites.}

^{TROUBLESHOOTING}

^{Problem: Multimers of plasmids are obtained after recombineering.}

^{[Steps 21, 32, and 57]}

^{Solution: Follow Steps 57-62 and perform selection using appropriate antibiotics after retransformation. Alternatively, choose a suitable restriction enzyme to linearize plasmids followed by religation and retransformation of the plasmids. This will eliminate multimers of plasmids.}

^{Problem: A high number of background colonies are found in uninduced controls following recombineering.}

^{[Steps 21, 32, and 57]}

^{Solution: Consider the following:}

^{1. One of the main reasons that background colonies are observed is incomplete digestion of plasmid template used for PCR. Always gel-purify the selection cassettes prior to PCR amplification. We use 1 µg of plasmid DNA for digestion in a 30-µL volume with 20 units of each restriction enzyme. The reaction is usually for 2 h at 37°C.}

^{2. To further eliminate the background, digest PCR products with DpnI (NEB). DpnI only cleaves DNA from dam+ strains, and plasmids prepared from commonly used E. coli (DH5 and DH10B) are sensitive to DpnI digestion.}

^{3. Test the PCR-amplified cassette DNA by electroporation into electrocompetent E. coli to ensure that no background colony is obtained.}

^{Problem: No drug-resistant colonies are observed after antibiotic selection.}

^{[Steps 21, 32, 46, and 57]}

^{Solution: Consider the following:}

^{1. Check that the BAC clones still retain recombineering competence by testing for resistance to Hygro. If BAC clones are now sensitive to Hygro, repeat Steps 6-11.}

^{2. For Step 46, the problem could also be due to the rigors of double selection. In this case, perform single-drug selection instead of double-antibiotic selection. Remove drug selection for BAC or pSim plasmid when selecting for recombinants. Check recombinants by restriction digestion to confirm that the right clones are obtained.}

^{3. This problem could also be due to polymorphisms in the homology arms of the PCR-amplified selection/retrieval cassettes. In our laboratory, we find that the 50- to 80-nucleotide homology in the long oligos is generally sufficient for high-efficiency recombineering. The two homology arms flanking the selection cassettes should not have stretches of more than five similar nucleotides between them.}

^{Problem: Only empty vectors are obtained after the retrieval step.}

^{[Step 32]}

^{Solution: The retrieval vector is recircularizing due to microhomologies in the retrieval vector homology arms. Redesign the primers for generating the homology arms for the retrieval vector and repeat retrieval Steps 24-31.}

^DISCUSSION

^{Phage-based E. coli homologous recombination systems have been extensively developed and optimized such that recombineering is now the preferred technique for carrying out genetic modifications in either the chromosome or plasmids (Zhang et al. 1998 ; Datsenko and Wanner 2000 ; Yu et al. 2000 ; Liu et al. 2003 ; Wang et al. 2006 ; Chan et al. 2007 ). Recombineering is a highly efficient and precise process that circumvents the problems of traditional genetic engineering by eliminating the need to locate specific restriction enzyme sites. Conventional or conditional gene knockouts, point mutations, amplifications, and deletions can be readily introduced into the genomic DNA of interest using recombineering, thus greatly reducing the time required to generate sophisticated DNA constructs. In contrast to our previous protocol (Liu et al. 2003 ), this protocol uses a pSim plasmid, which carries the phage homologous recombination genes (Exo , Bet , and Gam ). Therefore, BAC cells can be readily made recombineering-competent by transformation with the pSim plasmid. The protocol described here also allows the generation of a flexible allele that can function as a reporter, cko, or null allele. In addition, the various recombineering steps are optimized such that the entire targeting construct process can be performed in 96-well plates (Chan et al. 2007 ). This robust recombineering protocol is thus suitable for generating targeting constructs in a rapid and highly efficient manner.}

^{ACKNOWLEDGMENTS}

^{This work was supported by the Wellcome Trust.}

^REFERENCES