i’ve been having trouble with microinjection. i am able to generate F1 progeny, but they never transmit to the F2 generation. the transgene does not appear to inhibit anything to do with fertility. is there something that may be hindering my worms ability to transmit the gene that i’m working with?
im injecting my transgene at 15 ng/ul, the marker at 10 ng/ul and i bring the total concentration of DNA to 100 ng/ul with N2 genomic DNA. i have been injecting mainly late l4s and young adults. i dont know why im having so much trouble.
what is your marker? i’m guessing it is some GFP thing since 10 ng/ul is pretty low for pRF4.
anyhow, if control injection of marker plus N2 DNA does not give transmitting lines, then it is not your transgene causing the problem.
my guess is that you just aren’t getting good array formation.
you could try injecting your transgene along with about 150 ug/ml of pRF4.
or you could just try increasing the concentration of N2 DNA. when it is at a good concentration you won’t get many F1 transgenics, but ones that you do get will be likely to transmit to the F2.
if you really want good germline expression you might want to do MosCI.
why are you injecting into L4s? one day old adults will give you plenty of transgenic progeny and they are much easier to inject.
older worms did the trick. my transgene was a bit toxic so i had to reduce its concentration to 2.5 ng/mL. i co-injected with sur-5::gfp and i have plenty of green babies.