- Why I am not getting Rol or drug resistant animals from my injections?
Assuming you are experienced at injecting, the most likely cause of the problem is toxicity of the construct. In particular, I have had severe toxicity issues with inserts that contain both a driver and a reporter [example: mec-4p::tetR-L-QF::tbb-2 3′ UTR::tetO 7x::GFP-C1::his-58 ]. Injecting this DNA at 50 ng/ul leads to lots of very brightly fluorescent dead eggs, but no Rol progeny. To integrate this construct, I grew the landing site strain on doxycycline (1ng/ml) and injected less concentrated insert DNA mixed with carrier DNA (10 ng/ul insert construct, 100 ng/ ul pBluescript). I have observed similar problems with GAL4 driver/reporter lines, and in this case adding doxycycline won’t help. In the case of rRMCE, injections of a few plasmids designed to express a toxic compound (e.g. expressing TeTxLC) have failed to yield HygR animals and in these cases the injection plates had lots of very brightly fluorescent dead eggs. In such cases, the best option is to dilute the DNA. I have gotten successful integration events at reasonable frequency injecting as little as 5 ng/ul of insert plasmid supplemented with 150 ng/ul of pBluescript. The key is to get DNA containing the insert plasmid to the F1 germline because it is in the F1 germline that most of the integration events occur. I have had only very rare problems creating transgenes that carry only a driver, only a reporter, or contain inserts that harbor a worm promoter directly driving a reporter.
2. The efficiency of RMCE is lower than your paper claims? What is going on?
The efficiency of RMCE is highly temperature dependent. Perform your post-injection incubations (first 2 generations) at 25° C. In my experience even dropping to 22.5 to 23° C (RT in my lab) will lower the efficiency significantly. I also recommend using a PB wash step in your protocol for preparing miniprep DNA for injection. My lab uses Qiagen miniprep columns for DNA purification from E. coli.
3. The landing site strains are supposed to express ubiquitous nls-GFP, but I do not see that signal?
The rpl-28 promoter that drives nls-GFP in the landing sites is strongly down-regulated in adults. It can be very difficult to see the nls-GFP signal especially in adults after they start laying eggs. The nls-GFP signal is highest in L2, L3 and L4 animals. It is easiest to see in the intestine. I can detect the signal in L1s, and very easily detect the signal in L2 through L4 animals on the high power of a compound dissecting scope. The nls-GFP signal is also diffiuclt to see in starved animals of all stages. The mNeonGreen signal from bqSi711 (mex-5p FLP D5 sl2 mNG) or similar cassettes in single component landing sites (e.g. jsTi1493 IV) is just the opposite. This germline signal is difficult to detect in the dissecting scope until early adult, though it is detectable in L4s. On the compound scope one can detect the cytosolic mNG signal in the germline in all larval stages. In case you are wondering, I have a Lieca MZ16F equipped with a rotating tourette and a 5X PlanApo 0.5 NA LWD (long working distance) lens, and a Sola LED light engine light source with an optical cable.
4. Do I need to perform molecular analysis to confirm the structure of my insert?
In my initial characterization of the technique I performed long range PCR across over 40 distinct insertions and found no rearrangements, deletions or insertions in any transgene inserted at jsTi1453, jsTi1490 or jsTi1492 (Nonet, 2020). I have isolated a few unexpected insertions, but all of these were spontaneous non-Rol animals that appeared in the absence of heat shock. I do not recommend picking those animals even though in most cases these animal are the result of proper excision of the loxP SEC. My usual practice is to assume the insertions are correct if I am getting the expected expression pattern, behavior or phenotype for a transgene. However, if I get unexpected results I perform long range PCR across the insertion and either restriction mapping or sequencing of the resulting product to characterize the insertion locus. Since the spring of 2022 one can economically ($15) sequence the entire insert using nanoPore sequencing at plasmidsaurus. I simply amplify the locus using a long range PCR protocol, column purify, and sequence the product. Slight background is tolerated in my experience.
5. I have drug resistant animals (rRMCE) or Rol animals (RMCE), but I cannot homozygose the insertion. What is going on?
Such lines could either be unusual extrachromosomal arrays or insertions into an essential locus via non-FLP mediated integration. If you have such a case, please contact me. I would like to get a hold of such lines. I would like to document such cases and determine if these are insertions or unusual and unexpect arrays.
Send me your questions by email and I will add them to this FAQ list.