1) Pick out an appropriate Mos1 allele on wormbase. We picked Mos1 insertions located between genes that were expressed based on EST data. You might have other/better criteria.
2) Order the allele from the NemaGENETAG consortium.
3) Cross Mos1 insert into appropriate background. We used unc-119(ed3). You may want to use another rescue marker.
4) Build cloning vector with approx. 3 kb homology to genomic insertion sites (centered on Mos1 insertion site).
5) Test for insertion and expression.
Please let us know if you notice obvous differences between any two sites.
Following Mos1 inserts
|Site||Forward oligo||Reverse oligo||PCR fragment (no Mos1)||PCR fragment (w Mos1)||PCR w forward oligo and oJL103|
|ttTi5605||TTTCTCAGTTGTGATACGGTTTTT||CGCTACTTACCGGAAACCAA||0.6 kb||1.9 kb||0.6 kb|
|cxTi10882||ACCTTCCAATCCGCCATATCC||TCCCCATTTCACCAGAGAAC||0.9 kb||2.2 kb||1 kb|
|cxTi10816||GGCATTTGATGCGATGAGTA||CAGTAGGGCCCGTGTAAAAA||0.4 kb||1.6 kb||not tested|
|ttTi4348||CTCTCGTCTCTCCACGATTTACAC||GGCGCAAGAACTGCGATTAG||0.5 kb||1.8 kb||not tested|
|ttTi4391||TCCCTACAGTATCCCTACAGC||ACCCGTTCAGAATATACCCAG||0.4 kb||1.7 kb||not tested|
|ttTi14024||TCGAACACGTACATGACGACTG||CAACGTGCCAGTTGTTGACT||0.3 kb||1.6 kb||not tested|
Hmm – it’s the most frequent question but also the most difficult to answer. Although many labs have told us they have good luck with the technique, then there are many things that could make insertions difficult: Your transgene could be toxic, the DNA you are injecting is not clean enough, you have limited experience with injections, you don’t like injecting into unc-119 animals…
First: Are you following the protocol we published? We are sorry to say this but most often when the technique is not working it is because one or more steps of the protocol have been changed. There are undoubtedly still aspects that can be improved (please drop us a line if you make improvements!) but for the most part the protocol works pretty well. One key part of the protocol for direct injections is to not pick F1 animals off the injection plates onto other plates. Some are used to doing this when they make “regular” transgenic lines and have kept that habit. Although we don’t have conclusive experimental data ourselves it really seems that the direct injection does not work (or at least works very poorly) if you pick rescued F1 animals. We have now heard from several labs that tried picking rescued F1 worms and it has not worked for them. If they don’t pick F1 animals, then the protocol works in their hands, too. It’s possible that MosSCI inserts often come from animals that are not phenotypically rescued and you would then throw these events out by picking F1 rescued animals.
Let all the injection plates (which each started with one individual injected animal) starve out completely. Don’t distribute F1s – forget about the plates until starved. It’s ok to put the plates at 25C. Actually, it appears that it may improve insertion frequency. Once the plates are starved, then screen the animals by fluorescence for animals that move like wild type animals but lack fluorescent markers. Then clone out 8-10 individual, non-fluorescent animals to individual plates. Several of these plates will typically breed true and have homozygous animals.
Second: It seems like the number of transformed F1 progeny per injected animal is the most important parameter for successful insertions. We get between 10-15 visibly rescued F1 animals per injected animal. Injecting young adults kept at 15C increases the survival rate and number of transformed F1 progeny. Also, growing animals on comamonas bacteria increases the brood size and health of unc-119 animals. Placing the animals at 25C after injection seems to be fine, though, and speeds up the process considerably.
If you are getting a good number of rescued F1s but no insertions, then we suggest you try the positive control plasmid pCFJ68 (Punc-122::GFP) which we’ve used to characterize the insertions sites with.
It’s not yet clear how well the different promoters express in the germline. If your promoter has a lot of complex sequences (simple or complex repeats) it may be disrupting the DNA repair machinery. See if there is anything on the germline page that can help you out.
Also, did you wait for 3-5 generations after getting the insert? Hereditary silencing from the injection carries over for several generations but sometimes goes away. Re-check you inserts every couple of generations.
For a more thorough characterization of silenced MosSCI inserts see this paper from the Mello lab.
Jonathan Hodgkin has determined that MosSCI inserts should have a new designation “Si”. So, transgenes integrated by MosSCI are designated by italicized names consisting of the laboratory allele prefix, the two letters Si, and a number.
For example, inserts from the Jorgensen lab will be named oxSi31[Punc-122::GFP cb-unc-119(+)] II.
So far, we have not detected any “off-site” insertions in > 40 inserts we tested.