{"id":37,"date":"2013-02-20T14:48:49","date_gmt":"2013-02-20T14:48:49","guid":{"rendered":"http:\/\/wormbuilder.org\/old\/?page_id=37"},"modified":"2014-01-31T22:18:04","modified_gmt":"2014-02-01T04:18:04","slug":"protocol","status":"publish","type":"page","link":"https:\/\/wormbuilder.org\/old\/?page_id=37","title":{"rendered":"Protocol"},"content":{"rendered":"

Quick protocol<\/h3>\nStep 1<\/span>Make targeting vector containing your transgene<\/strong><\/p>\n

Each insertion site has a specific vector and an injection strain that is compatible with that targeting vector. See plasmids and injection strains for info.<\/em><\/p>\nStep 2<\/span>Maintain injection strain on HB101 bacteria at 20 degrees<\/strong><\/p>\n

<\/strong>Healthy worms are much easier to inject and result in higher insertion frequencies. Maintain\u00a0unc-119<\/em>\u00a0strains at 15C or 20C.<\/em><\/p>\nStep 3<\/span>Make injection mix<\/strong><\/p>\n

<\/strong>Use 10-50 ng\/ul of targeting vector and add co-injection markers (pCFJ601 @ 50 ng\/ul, pMA122 (optional) @ 10 ng\/ul, pGH8 @ 10 ng\/ul, pCFJ90 @ 2.5 ng\/ul, pCFJ104 @ 5 ng\/ul). If necessary, add DNA ladder for a total concentration of 100 ng\/ul. High-quality DNA may improve insertion frequency<\/em><\/p>\nStep 4<\/span>Inject animals<\/strong><\/p>\n

<\/strong>Place one, two or three animals on each seeded plate. Put the injected animals at 25C until they starve out completely (~ 1 week).<\/em><\/p>\nStep 5<\/span>Heat-shock injected animals (optional)<\/strong>
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If you added the negative selection marker pMA122, you can kill (most) array animals by a two hour heat-shock at 34C in an air incubator. Array animals are dead approximately 4 hours after heat-shock.<\/em><\/p>\nStep 6<\/span>Screen for mosSCI insertions<\/strong><\/p>\n

<\/strong>Screen plates for animals that move like wildtype worms but do not have any of the red co-injection markers. Pick a couple of non-fluorescent, rescued animals to a new plate.<\/em><\/p>\nStep 7<\/span>Single and characterize insertion<\/strong><\/p>\n

<\/strong>Homozygose the insertion starting from a single, clonal animal. Characterize and verify insert in enough detail for your particular experiment (this ranges from just verifying fluorescence of a reporter strain to more elaborate molecular methods to verify that the full transgene was inserted).<\/em><\/p>\n

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Detailed protocol<\/h3>\n

Background<\/h5>\n

The basic principle of Mos1 mediated Single Copy gene Insertion (MosSCI) is based on the mosTIC technique developed by Roberts & Bessereau (2007).\u00a0 We\u2019ve added selection markers to facilitate insertions, selected standardized genomic locations for insertions and generated a number of cloning vectors for sub-cloning or Gateway cloning.\u00a0We mainly selected Mos1 insertion alleles in intergenic regions between the 3\u2019 regions of two genes facing each other. The sites were selected to avoid disrupting predicted genes and promoters; however the gene predictions are continuously updated so make sure we didn’t pick a site in the middle of your favorite gene. We have not detected any overt phenotypes in strains carrying either the Mos1 insertion or genes inserted by MosSCI.\u00a0However, please keep in mind that we have not done extensive cellular or behavioral characterization of strains carrying insertions at these loci. Most insertions will be full length single copies. But some will be duplicate insertions, will contain small deletions or insertions or will have secondary Mos1 hops in the background. We suggest treating each insert as you would treat separate alleles.<\/p>\n

Gene insertions are generated at the targeted locus by repair of the double stranded break after Mos1 excision. Mos1 excision is induced by expression of Mos1 transposase. The break can be repaired by homologous recombination between the broken ends and sequences on an extrachromosomal array\u00a0 (see Figure 1, below). For efficient homologous recombination to occur we used 1.5 kb of homologous sequence to each side of the Mos1 insert.
\nWe have developed two complementary approaches to generate insertions. 1) Direct insertions 2) Selection based insertions. Both approaches are based on a using\u00a0cb-unc-119<\/em>\u00a0as the positive selection marker to identify inserts. This requires that transgenic lines are made in the\u00a0unc-119\u00a0background. In Erik’s lab we exclusively use the direct insertion protocol with the negative\u00a0peel-1<\/em>\u00a0selection.<\/p>\n

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Direct injection vs Selection-based insertion<\/h5>\n
Direct insertions<\/h6>\n
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Mos1 transposase is expressed under the\u00a0eft-3<\/i>\u00a0promoter and the Mos1 transposon is excised in the injected animal. Insertions are identified visually in the F2-F3 progeny of injected animals based on\u00a0unc-119<\/i>\u00a0rescue but the absence of fluorescent co-injection markers. With the\u00a0peel-1<\/i>\u00a0selection this screening can be done without a fluorescent microscope with approximately 10% false positive rate (you may need to optimize the negative selection – in our lab it only works in the incubator that has an internal fan). This method has the advantage of being quick (approximately a week) but requires more injections than the heat-shock based insertion protocol.<\/p>\n

Selection based insertion<\/b><\/h6>\n

For this method, we rely on two selection markers to recover integration events: A) Co-integration of a\u00a0cb-unc-119(+)<\/i>\u00a0rescue fragment provides a positive selection marker for identifying extrachromosomal and integrated transgenes. B) A temperature-sensitive\u00a0twk-18<\/i>\u00a0construct provides negative selection against the extrachromosomal array thus distinguishing integrations from extrachromosomal arrays (Kunkel et al., 2000). The negative selection is achieved by expressing a dominant, temperature sensitive K+ channel in the body wall muscles. Worms move normally at 15\u00b0C, allowing the extrachromosomal array to be propagated. When shifted to 25\u00b0C, the worms carrying the extrachromosomal array are paralyzed, allowing for selection of integration events. This method is slower (approximately a month) and requires more hands-on time as well as reagents. It has the advantage that it can be easily scaled and may therefore be preferable for large or for toxic transgenes.\u00a0Please note that we do not use the selection based method in the lab anymore.<\/p>\n<\/div>\n

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Direct injection protocol<\/h4>\n
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\"Direct<\/a><\/dt>\n
Direct insertion protocol<\/dd>\n<\/dl>\n<\/div>\n
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Preparation for experiments<\/b><\/span><\/h5>\n

To generate transgenic animals by MosSCI the transgene needs to be inserted into vectors that regions complementary to the genomic target site and the\u00a0cb-unc-119<\/i>\u00a0rescue fragment. See a description of the reagents\u00a0to find the appropriate vector.
\nWe have routinely injected miniprep quality DNA. However,\u00a0Morris Maduro<\/a>\u00a0has noticed that higher quality DNA results in more transformed F1 animals and it is therefore likely to improve insertion frequency. We recommend using midi-prep quality DNA for the co-injection markers (since you will only make those vectors once) and miniprep DNA for the targeting plasmid, with an additional ethanol precipitation of the miniprep DNA.<\/p>\n<\/div>\n

Direct insertions rely on insertion of transgenes in the germline of the injected P0 animal. Expression of the Mos1 transposase is achieved in the germline under the\u00a0eft-3<\/i>\u00a0promoter. Insertions can be identified in the F2-F3 progeny based on 1)\u00a0unc-119<\/i>\u00a0rescue, 2) survival of heat-shock with the\u00a0peel-1<\/i>\u00a0toxin, and 3) absence of co-injection markers.<\/p>\n

A week before injection you will need to pick injection strain animals for injection. After injection it takes approximately 7 days before you can screen for inserts. There are few experimental manipulations in this waiting period. In our experience, the frequency of successful insertions will depend on several factors: the purity of the injected DNA, the stage of animals you inject, how many rescued progeny you get from each injection, the structure of your transgene, and if your transgene is toxic. In our hands, we routinely get insertions from > one in every ten injected animals that have rescued F1 progeny. Of these, 60 % contain full, single copy inserts. In initial experiments we suggest you inject approximately 50 animals to ensure a reasonable success rate.<\/p>\n

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Materials and equipment<\/b><\/span><\/h5>\n

\u2022 Standard injection setup: inverted microscope, injector and micromanipulator.
\n\u2022 Negative selection marker: pMA122
\n\u2022 Fluorescence dissection microscope with mCherry filter set and shutter controlled by foot peddle.
\n\u2022 OP50 and HB101 seeded NGM plates (small)
\n\u2022 20\u00b0 incubator
\n\u2022 25\u00b0 incubator
\n\u2022 Co-injection plasmids: pGH8, pCFJ90, pCFJ104
\n\u2022 Mos1 transposase plasmid: pCFJ601
\n\u2022 Injection strains<\/p>\n<\/div>\n

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A week before injections<\/b><\/span><\/h5>\nStep 1<\/span>Maintain unc-119<\/em> animals from the injection strain on HB101 bacteria at 20\u00b0C. On HB101 plates, the unc-119<\/em> animals can move around better than OP50 so we pick a couple of adult worms to a few plates every day. That way the progeny is healthy and unstarved approximately a week later. You can also distribue eggs or L1 animals around a plate for faster propagation but we have better luck with injection animals that have not been starved and are healthy. The injection strains are distributed with an extra-chromosomal rescuing array for easier freezing and distribution but you should\u00a0inject into unc-119<\/em> animals.<\/p>\n<\/div>\n
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Day 0 – Injection<\/b><\/span><\/h5>\nStep 2<\/span>Make up injection mix of:<\/p>\n\n\n\n\n\n\n\n\n\n\n
\u00a0Plasmid<\/b><\/td>\n\u00a0Description<\/b><\/td>\n\u00a0Final concentration<\/b><\/td>\n<\/tr>\n
\u00a0pCFJ601<\/td>\n\u00a0\u00a0Peft-3<\/em>::transposase<\/td>\n\u00a050 ng\/ul<\/td>\n<\/tr>\n
<\/td>\n\u00a0Transgene in targeting vector<\/td>\n10 – 50 ng\/ul<\/td>\n<\/tr>\n
pMA122<\/td>\nPhsp<\/em>::peel-1<\/em><\/td>\n10 ng\/ul<\/td>\n<\/tr>\n
\u00a0pGH8<\/td>\n\u00a0Prab-3<\/em>::mCherry (Pan-neuronal)<\/td>\n\u00a010 ng\/ul<\/td>\n<\/tr>\n
\u00a0pCFJ90<\/td>\n\u00a0Pmyo-2<\/em>::mCherry (pharynx muscle)<\/td>\n\u00a02.5 ng\/ul<\/td>\n<\/tr>\n
\u00a0pCFJ104<\/td>\n\u00a0Pmyo-3<\/em>::mCherry (body muscle)<\/td>\n\u00a05 ng\/ul<\/td>\n<\/tr>\n
<\/td>\n\u00a0DNA ladder<\/td>\n\u00a0Add to total concentration 100 ng\/ul<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Note: We make a 2x stock of everything except the targeting vector and the DNA ladder. That makes it much easier to make several injection mixes.
\nSpin all DNA preps at highest speed on table-top centrifuge before making injection mix. Spin injection mix again immediately before injection.<\/p>\n<\/div>\n

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