Troubles rescuing with fosmids?

Has anybody had problems with rescuing a mutant phenotype with a fosmid? I have been trying to rescue mutants of our gene-of-interest by microinjecting a rescuing fosmid but I encounter very low transmission of the transgene, lethality and partial rescue at best. I have diluted the fosmid to a very low concentration and even cleaned-up the DNA with phenol:chloroform/Sodium acetate ethanol precipitation. Any help would be greatly appreciated!!!

I am trying to rescue with fosmids and having trouble too. Did you ever resolve your problem?


I have got a fully rescue of a mutant with a fosmid construct, but it is my only experience of rescue experiment . However, I am injecting fosmids on a daily basis and the optimal concentration for fosmid injection is between 1 and 20 ng per microlitres final concentration in the mixture of injection with pRF4 at 100 ng per microlitres. Some of the fosmid could be toxic and in that cases, it happened that I had to dilute 100 times to get transmitted lines. most of our transgenic lines exhibit a variable transmitted rate going from very bad 5% to very good 80 or more %.

that is what we are usually doing here in the lab. Hope it helps …