Worm Breeder's Gazette 13(2): 13 (February 1, 1994)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The following should be useful to people that are using the Green Fluorescent Protein (GFP) as a marker (see WBG 13 #1 p. 19)
I. Vectors
A. pGFP10 .1.This is the original GFP plasmid. People using it should not cut out a restriction fragment containing the entire coding sequence. There appears to be something upstream of the coding region that poisons expression. Instead PCR should be used to obtain the sequence.
B. Worm vectors. The modified Fire vectors described in the previous Newsletter contribution lack the Kpn I fragment with the SV40 nuclear localization signal. We are in the process of making vectors containing this fragment, but they are not ready yet.
II. Photobleachinq
A. Effect of Anesthetics. we had originally found that GFP expressed in C. elegans photobleached rapidly when illuminated with light using a 395-440 nm excitation filter (photobleaching with the 450-490 nm excitation filter normally used for FITC was not as rapid). We have now found that the very rapid photobleaching resulted from the use of sodium azide as an anesthetic. The fluorescence is much more long-lived without the azide. Unfortunately, in preliminary experiments phenoxypropanol seemed to eliminate the fluorescence entirely. We have not tried CO(2) as an anesthetic. This fact that azide causes photobleaching may be a useful tool.
B. Low Wavelength Excitation. We obtained an interesting result when animals were illuminated with 340-390 nm light (the excitation from the standard DAPI filter set). The green fluorescence rapidly disappeared, but when the animals were subsequently examined with light of the two higher wavelengths (see IIA), the green fluorescence remained. Similarly if the animals were first viewed for a few minutes with light of the higher wavelengths (and showed fluorescing cells) and then examined with the DAPI filter set, no fluorescence was seen. This suggests that there may be two molecular species produced in the animals. Interestingly, the fluorescence at the higher wavelengths seems to be stronger after the 340-390 nm excitation or just a minute or so of excitation at the higher wavelengths.
III. Fixation. As other people begin to use GFP, we are learning some interesting things about its properties. One of these was discovered by Shengxian Wang and Tulle Hazelrigg using GFP in Drosophila. They found GFP fluorescence survives formaldehyde fixation (subsequently I learned that there were unpublished results demonstrating that the native protein from jellyfish could also fluoresce after formaldehyde and glutaraldehyde fixation). I have confirmed that formaldehyde fixation does not abolish GFP fluorescence in C. elegans using the uIs4 animals. So fixed as well as living tissues can be examined.