Has anyone tried co-bombarding c.elegans with two plasmids (one carrying the unc-119 rescuing fragment + one carrying the rescuing gene fragment)? If so, what ratio of plasmids were used with successful results?
Bombarding with two seperate plasmids definitely works. I refer you to a previous thread where this was discussed.
I used equal amounts of gfp reporter plasmid and the unc-119 rescuing plasmid pMM016 (50 ug of each, as I recall). Roughly half of the resulting unc-119-rescued transgenic lines gave gfp expression; I don’t know how that compares to when the markers are cloned onto a single plasmid.
won’t using two separate plasmids allow the unc-119 rescuing transgene and GFP expression transgene to segregate independently?? this might make propagating the strain tricky…
won't using two separate plasmids allow the unc-119 rescuing transgene and GFP expression transgene to segregate independently?? this might make propagating the strain tricky...In every case I've heard of, when a transgenic line is recovered that shows expression from multiple co-injected or co-bombarded constructs, the line contains a single transgene combining the different constructs, and they therefore segregate together. Transgenes generated by co-bombardment or even by co-injection do not necessarily contain everything that was co-bombarded or co-injected, but those that do appear to do so fairly stably.
Thank you for your reply. I do not have access on the article online, so I will have to go to the school library to look for the book. The protocol I have says to use 7ug of plasmid to bombard (referring to a single plasmid bombardment). I tried using 3.5ug of each plasmid, when I mixed two. I have not seen any unc rescued worms yet, so I am concerned my concentration was too low.
Since you tried 50ug of each, I will increase my concentration of each next time. Thanks!
I used 100 ug of total DNA, but I was preparing microparticles for use with a Helios Gene Gun. In that protocol, 15 mg of microparticles are used to coat the inside of a piece of tubing approximately one yard (36 inches) long. This tubing, when prepared, is cut into half-inch lengths that are used as cartridges within the gene gun, so even if none of the DNA was lost by not adhering to the microparticles or on microparticles that didn’t coat the tubing, each shot would be using less than 1.5 ug DNA. I wouldn’t compare your 7 ug directly to my 100 ug unless we were using the same system.