1. User-defined synthetic oligonucleotides.
2. pENTR-NLS-G-FN. This carries a nuclear localization sequence (NLS), which is crucial for the Drosophila RNA injection method, and a 633bp fragment in place of a zinc finger coding sequence.
3. pCS2-DestA. A plasmid modified from pCS2, a multipurpose expression vector. It introduces an SP6 promoter and the SV40 late polyadenylation site.
These plasmids and their full nucleotide sequences can be obtained from the Carroll Lab.
4. Aquagenomic DNA Extraction Reagent (MultiTarget Pharmaceuticals, Salt Lake City , UT ; http:// www.aquaplasmid.com ) for preparing genomic DNA from single adult flies. Your favorite DNA extraction protocol is fine, as well.
5. E. coli strains: DB3.1 (Invitrogen) for propagating pCS2-DEST or any other DEST vectors (required to provide resistance to the product of the ccdB gene); DH5 alpha .
6. LR Clonase II Enzyme Mix (Invitrogen).
7. In vitro transcription kit with SP6 RNA polymerase. We have used both AmpliCap SP6 High-yield Message Maker (Epicentre, Madison , WI ) and mMessage mMachine SP6 (Ambion, Austin , TX ) with good results.
8. Standard molecular biology supplies and a Drosophila injection setup.
1. Choose a gene to be targeted and search it for sequences that look like good ZF binding sites in proper relative position. The search can be performed at either of two websites designed for the purpose: one maintained by Carlos Barbas' lab (38) , http://zincfingertools.org , and one by the Zinc Finger Consortium, http://www.zincfingers.org . The features of a promising site for 3-finger ZFNs are inverted 9-bp sequences made up of triplets for which good fingers exist separated by 6 bp. In some instances the 6-bp spacer will include or overlap a restriction enzyme recognition sequence, which allows molecular screening for NHEJ products, but this will not always be possible. Additional details are given in our Nature Protocols paper (Carroll et al, 2006)
2. Design amino acid sequences of the ZFs that will bind each of the component 9-bp sites Again, the Nature Protocols paper discusses this extensively.
3 Synthesize long oligonucleotides corresponding to the designed coding sequences, and combine them by PCR (Be sure not to create a NotI site) . The product for each 3-finger subsite should be 283 bp long, if our exact protocol is followed. The long oligos can be HPLC purified to remove incorrect products, but we find it sufficient to just sequence a few clones to identify one with the correct insert.
4. If necessary, reamplify the 283-bp PCR product.
5. Ligate into pENTR-NLS-G-FN in place of the existing sequence. This requires cleaving vector and PCR product with NdeI and SpeI, recovering the desired fragments, and ligation.
6. Transform into competent E. coli cells - e.g., DH5? - and select on plates containing kanamycin (50 µg/ml).
7. Pick individual colonies and screen for the correct inserts - e.g., by colony PCR using one primer located in the new insert, and another complementary to vector sequences. We use pENT-F, TTTAT AATGC CAACT TTGTA CAAA, which binds to pENTR, and AB-F3-R, TGACT AGTTG CTTCT GTCTT AAATG GATTT TGG, which binds to the 3' most constant sequence in the fingers. These will not recognize the insert in the cloning vector, but will recognize any insert constructed using our published protocol and sequence. Ultimately, the inserts should be verified by sequencing.
8. Purify the verified plasmid DNAs - e.g., by a mini-prep procedure using a Qiagen kit. Large preps are not necessary at this stage. We will refer to these constructs as pENT-NLS-ZFNa and pENT-NLS-ZFNb
1. Run Clonase reactions, as described by Invitrogen, using your pENTR-NLS-ZFNa and -ZFNb constructs and the pCS2-DEST vector.
2. Transform DH5 a , plate on Amp plates, verify the resulting clones, and make highly purified DNA, such as a Qiagen maxi prep. Although the Clonase reaction is clean and reliable, occasional artifacts do occur. We normally verify by digestion, and by sequencing. These constructs are pCS2-ZFNa and pCS2-ZFNb
3. Linearize 2-4 µg of each plasmid by digestion with Not I, which cuts distal to the 3'-UTR.
Transcribe the pCS2-ZFNa and pCS2-ZFNb DNAs separately using the AmpliCap or mMessage mMaker kit (or similar), following the protocol with the kit. Examine the resulting RNAs by agarose gel electrophoresis to ensure that they are full length. The RNA will require concentration. We have used both ethanol and sodium acetate precipitations, bringing the reaction up in ~8µl of ddH 2 O
If you want to include a donor DNA, the nature of this component will depend entirely on the gene being targeted and the alteration desired. It must have homology with the target on both sides of the ZFN cut site, and the ZFN site in the donor should be modified to prevent its cleavage. (This adjustment may not be necessary, but seems prudent.) Although we have not tried extensive donor configurations, we expect any configuration that has been shown to work in a Rong and Golic protocol should work in this protocol. You DO NOT need any special meganuclease sites or FRTs. In fact it is probably best to just design your donor in a small vector, such as Bluescript or pGEM, so that the donor makes up a large percentage of the plasmid. Make highly purified, concentrated DNA, i.e. 1000-2000µg/ml. The plasmid should be injected uncut.
1. Prepare an injection mix that has 350-800 µg/ml of each RNA. If gene replacement is desired, include the donor DNA in a circular plasmid at 1500 µg/ml. These concentrations may require optimization for your particular constructs, but we have had reduced success with higher concentrations of donor. In our experience, higher concentrations of RNA result in fewer survivors, but a higher yield of mutants. We use an injection buffer of .1X PBS, and attempt to keep our workstation as RNAse free as possible.
2. Inject ~300 Drosophila embryos of an appropriate genotype for your experiment with this mix. In our hands, the injections are physically somewhat more difficult than DNA injections. After conversations with others who have injected RNA, we theorize this is due to RNA not completely redisolving after precipitation. We are working on this issue.
2. When adults eclose, cross them individually to mates designed to ease screening and recovery of new targeted mutations. Screening protocols will depend on your particular situation. Mutants frequently occur in large clusters, so one possibility is to cross your targeted animals to a stock carrying a deficiency over a balancer, screen the progeny that carry the deficiency for your phenotype, and collect the balanced siblings of positive animals, then testing those for mutations. Another possibility is to introduce a visible insert ( white or GFP ) into your donor.
Porteus, M. H., and Carroll, D. (2005) Gene targeting using zinc finger nucleases. Nat. Biotechnol. 23, 967-73.
Beumer, K., Bhattacharyya, G., Bibikova, M., Trautman, J. K., and Carroll, D. (2006) Efficient gene targeting in Drosophila with zinc finger nucleases. Genetics 172, 2391-403.
Bibikova, M., Beumer, K., Trautman, J. K., and Carroll, D. (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764.
Bibikova, M., Carroll, D., Segal, D. J., Trautman, J. K., Smith, J., Kim, Y.-G., and Chandrasegaran, S. (2001) Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol. Cell. Biol. 21, 289-97.
Bibikova, M., Golic, M., Golic, K. G., and Carroll, D. (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169-75.
Carroll, D., Morton, J. J., Beumer, K. J., and Segal, D. J. (2006) Design, construction and in vitro testing of zinc finger nucleases. Nature Protocols 1, 1329-41.
Gong, W. J., and Golic, K. G. (2003) Ends-out, or replacement, gene targeting in Drosophila . Proc. Natl. Acad. Sci. USA 100, 2556-61.
Wright, D. A., Thibodeau-Beganny, S., Sander, J. D., Wiinfrey, R. J., Hirsh, A. S., Eichtinger, M., Fu, F., Porteus, M. H., Dobbs, D., Voytas, D. F., and Joung, J. K. (2006) Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nature Protocols 1, 1637-1652.