It is clear that Tol2 transposons are working very well and efficiently in many labs; others use Sleeping Beauty transposons or I-SceI meganuclease.
It would also be useful to develop more transposon systems (perhaps PiggyBAC), which may have different target biases and would provide another system orthogonal to Tol2.
BAC transgenesis is being used by quite a few labs but seems to be relatively tricky and perhaps inefficient. Does anyone have major improvements (has anyone put Tol2 ends into a BAC)?
Two problems that seem to appear sometimes are variegation (i.e., contruct not expressed in only a subset of the cells that should express it) and silencing over generations. There seems to be a feeling that variegation happens with Tol2 transgenesis as well as DNA plasmid transgenesis; it's not clear whether silencing depends on the method of transgenesis.
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I would like to know how to get BAC transgenesis to work more efficiently perhaps in combination with the Tol2 system as Chi-Bin pointed out? How big can a piece of DNA be integrated by Tol2?
I have only made one BAC transgenic (and it subsequently became silenced -- arrgh!) but I got it after only screening about a half dozen or dozen potential founders. My BAC was from the CHORI-211 library in pTARBAC2.1. Although I've been told this vector doesn't have an I-SceI site, the map I had showed one so I co-injected the meganuclease -- I don't know if that helped. I will be trying again soon to get a non-silenced line...
-jim
I've rechecked the pTARBAC2.1 sequence and it has a near-match (the first 36 of 39 bases listed in the site given in the NEB catalog) for a different homing endonuclease, PI-Sce I, not I-Sce I as I had thought. But perhaps co-injection with PI-Sce I would improve transgenesis efficiency with BACs in this vector?
-jim
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