I want to make some transgenic lines by microinjection without using Rol or GFP as markers. I realize there are multiple options, but I wanted to focus my question here on the use of unc-119.
Although it is well-known for its use with microparticle bombardment, others have also used for microinjection the unc-119(ed3) + pDP#MM016 system (Maduro and Pilgrim, 1995).
My question: does the associated Daf phenotype in the unc-119(ed3) DP38 strain render it significantly less optimal for microinjection relative to the non-Daf HT1593 strain? For example, are DP38 animals Egl (causing lower broods) or less fertile compared to HT1593, such that I would expect to get fewer transgenics per injected animal?
I was wondering which strain I should order from the CGC for my purposes.
DP38 is Daf-d (dauer-defective) because unc-119 mutants are Daf-d and can suppress some Daf-c (dauer-constitutive) mutants. So DP38 would appear indistinguishable from HT1593 until it was rescued for unc-119. All unc-119 null mutants have reduced brood sizes, are partially Egl and are somewhat harder to inject than wild-types. There is some useful information on improving unc-119 injection on the Jorgensen lab’s web page on MosSCI (which involves injection of a mixture of plasmids into unc-119 mutants):
Mike Nonet sent us the following advice from his lab: "We have been growing the EG5003 and EG4322 strains on DA1877 Comamonas bacteria at 15C and under these conditions the strains grow larger, more healthy, have larger brood sizes and larger gonads for injections." In our own hands, we find that HB101 bacteria is preferable. The animals are healthier than on OP50 bacteria but the HB101 strain is much easier to grow and maintain.
As any background mutation (such as a Daf-c mutation) would probably not be desirable in a transgenic, it would be best to use HT1593 or any other outcrossed unc-119 mutant strain.
Thanks so much for the clarification on DP38 with respect to the Dauer phenotype. I understand now. Indeed, it sounds like HT1593 is what I want. I also appreciate you passing along the tips from the Jorgensen and Nonet labs. Now where can one get their hands on pDP#MM016?
