Worm Breeder's Gazette 13(1): 19 (October 1, 1993)
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.
We have developed a new way to look at gene expression in C. elegans (and other organisms) that utilizes an inherently fluorescent protein (the green-fluorescent protein; GFP) from the jellyfish Aequorea victoria. GFP fluoresces bright green when illuminated with blue light. We have found that this fluorescence does not depend upon any other component specific to A. victoria, so gfp can be used instead of lacZ, for example, to make gene expression fusions.
We have made a mec-7 gfpfusion using the mec-7 promoter, transformed C. elegans with this construct, and generated two integrated lines to examine GFP expression. Both lines (and the parental non-integrated strain) were fluorescent, but one insertion gave very strong fluorescence ( uIs4 ).Strong expression is seen in the four embryonic touch cells (the ALM and PLM cells) in uIs4 animals. Even the terminal branches of these neurons can be followed. Other cells also fluoresce, but less strongly (BDU, FLP, a few cells in the tail, and the AVM and PVM touch cells). Two additional cells in the tail also show fairly strong fluorescence: by the projection of their processes, these appear to be the ALN cells. The staining of the ALM, AVM and PVM (but not to as great an extent in the PLM cells) was dependent on mec-3 .These results are consistent with the previous expression pattern produced by this promoter [Hamelin et al., EMBO J. 11, 2885 (1992); Mitani et al. Development, in press] and seems to be equal to our most sensitive method (antibody staining). (The ALM and PLM cells are often displaced anteriorly in uIs4 animals, but not in the other strains; this defect is probably due to a secondary mutation or a mutation at the site of insertion.)
We have not completely optimized the method of viewing the GFP fluorescence. The excitation spectrum for native and recombinant GFP has a major peak at 395 nm and a minor peak at 470 nm, and the emission spectrum has a major peak at 509 nm with a shoulder at 540 nm. Because we found that 395 nm light causes a very rapid photobleaching that is not seen at 470 nm (the fluorescence bleaches, but slowly; there is recovery from photobleaching at both wavelengths), we have tended to use the higher exciting wavelength. The standard FITC filter sets provide the appropriate light. However, refinements can be made. For example, we find that it is better to use a long-pass emission filter (GFP looks green and the animals' autofluorescence is yellow) rather than a band-pass filter (both are green). (In preliminary observations with several of the flu strains we haven't seen any improvement. We haven't yet looked at clr-1 animals, but these would presumably help eliminate the problem of the autofluorescence.) Another improvement comes from using a xenon rather than a mercury lamp for fluorescence (the output dips at 470 nm with the mercury lamp, but not with the xenon lamp). We have not yet tried low-intensity-light video cameras (the autofluorescence may pose a problem here).
We have lots of ideas of how gfp might be used and imagine that other people will have many more. We think it should be possible 1) to examine gene expression and protein localization at various stages (and to see changes in expression, e.g. through cell division); 2) to examine the outgrowth and migration of cells in situ; 3) to look for mutants that change the pattern of expression [e.g., looking for revertants of the degeneration-causing mec-4 ( e1611 )mutation by mutating a mec-4 gfp; mec-4 ( e1611 )double and looking for the reappearance of fluorescing cells], 4) to mark cells for subsequent isolation and study (an experiment we hope to do soon with Shawn Lockery - who suggested the above title), and 5) to identify cells for laser ablations (the cells may also absorb more laser energy).
We have generated a set of plasmids that may be useful for C. elegans researchers. These are a pBluescript II KS (+) derivative ( TU#65 )containing a Kpn I - ECoR I fragment encoding GFP with an Age I site 5' to the translation start and a Bsm I site at the termination codon (suggested by Andy Fire) and gfp versions ( TU#60 - TU#63 )of the four C. elegans lacZ expression vectors ( pPD16 .43, pPD21 .28, pPD22 .04,and pPD22 .11,respectively) described by Fire et al., Gene 93, 189 (1990). If you are interested in obtaining any of these clones, please write (or FAX or email) your request (include your FAX number; we'd like to know what you are interested in doing, but that's not essential) to Marty Chalfie and he will FAX you the necessary Columbia papers to sign (they can be returned by FAX) and we will try to send out the clones immediately.