Worm Breeder's Gazette 15(5): 20 (February 1, 1999)

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.

Wild-Type Chromosomes Carrying GFP Markers

Mark Edgley1, Jun Kelly Liu2, Don Riddle1, Andy Fire2

1 Division of Biological Sciences, University of Missouri, Columbia, MO 65211
2 Carnegie Institution of Washington, Department of Embryology, 115 West University Parkway, Baltimore, MD 21210

Chromosomes carrying integrated green fluorescent protein (GFP) transgenes can be useful as integrated dominant markers for individual chromosomes, since the marker itself causes no phenotype that could interfere with viability, fertility or mating. Most existing GFP integrants were created for specific purposes, such as analyzing expression patterns of particular genes. The GFP expression in these strains is usually limited spatially and temporally, which can restrict the ease of detection. Moreover, these integrants have usually been generated with a transformation marker, such as rol-6 or dpy-20. We imagined that many investigators would find uses for wild-type chromosomes carrying mapped insertions of a GFP construct without any other transformation marker, especially if expression began early in development and persisted through the adult in enough tissues to make detection easy in a dissecting microscope with UV illumination.

We have made such a set of five strains, carrying a GFP-expressing element integrated into wild-type chromosomes. The strains and genotypes, as well as the initial mapping data, are listed in Table 1. These strains originated in screens designed to integrate the element into either the reciprocal translocation eT1 or the inversion mIn1[dpy-10] (formerly mC6). A GFP integrant on mIn1 was also generated in this screen (Edgley and Riddle, in preparation).

The integrated element began life as a three-construct extrachromosomal array, with GFP fused to promoters for pes-10, myo-2 and a gut-specific enhancer. The array didn't bear any other transformation marker, and the transformants were directly picked under the fluorescent dissecting scope. Worms carrying the array expressed: [1] cytoplasmic GFP in 4-60 cell stage embryos (pes-10), [2] diffuse nuclear GFP in gut precursors in embryos starting around 60 cell stage and persisting in the gut through larval and adult life, and [3] cytoplasmic GFP in the pharynx (myo-2) from mid-stage embryos throughout the animal's life.

The ccIs9753 insertion was generated by following standard integration methods using mIn1[dpy-10(e128)]/hlh-1(cc450) lin-31(n301)II animals, which carried the GFP array transmitting at about 50% frequency (ccEx9747). For the mIs insertions, young adult hermaphrodites of two stable transgenic stocks carrying this array (eT1(III;V); ccEx9747 or mIn1[dpy-10(e128)]/hlh-1(cc450) lin-31(n301)II; ccEx9747) were mutagenized by gamma irradiation with a Co60 source at 4500R. About 60 mutagenized animals of the former strain and 42 of the latter were plated on NG agar with food at 7-10 animals per plate, and 250 GFP-expressing F1 hermaphrodites for each strain were picked individually to microtiter wells (50 µl 1% OP50 in S medium per well). The plates were screened using a dissecting scope under UV illumination for individual wells in which most or all F2 animals expressed GFP (an F1 animal heterozygous for an insertion would segregate 75% GFP+ progeny; if such an animal also carried ccEx9747, segregation of GFP+ progeny could reach 100%). These wells were transferred by pipette onto NG agar plates seeded with OP50. Six F2 animals were subcloned from each promising population. In this way, four integrated lines were obtained in an mIn1 background, and two in an eT1 background. One insertion appeared to be on mIn1[dpy-10], and GFP appears to be a stable, dominant marker for this balancer chromosome. No integrations occurred within the translocated portions of eT1. The other five insertions were crossed three times with N2 animals and mapped to specific linkage groups. mIs12 III was isolated in the eT1 screen, but was inserted into the untranslocated left arm of eT1 III. This insertion has been crossed onto a wild-type chromosome III.

These stocks have been lodged with the CGC for distribution. The pes-10::GFP signal is very faint and invisible under the fluorescent dissecting scope. The gut-specific GFP is visible in embryos in the dissecting scope at 25-50X as a green smudge, and is variable in adults but still easily seen. The pharyngeal signal is very bright (except in mIs13, where it is somewhat attenuated) and easily seen. The strains are very easy to use and should provide many hours of productive entertainment and viewing pleasure. We would like to welcome any further mapping data and male mating efficiency data from the community about these integrants in your future uses of them.

Table 1






I (dpy-5 unc-54)14/2147/6219/420/814/58
II (dpy-10 unc-52)5/1318/209/1225/4236/61
III (unc-32 dpy-18)33/3836/4012/7360/8132/62
IV (unc-17 dpy-4)11/179/2024/3629/3934/54
V (unc-60 dpy-11)5/3830/3911/1528/4743/62
Linkage Summary:VIVIIIII

Table 1. Summary of mapping data for each integrant. Homozygous GFP+ hermaphrodites for each strain were crossed to N2 males. F1 males were crossed to each Dpy Unc marked strain. The resulting GFP+ hermaphrodites were cloned and their Dpy Unc progeny were scored for the presence or absence of GFP. The numbers in each column represent the number of GFP+ Dpy Unc animals over the total number of Dpy Unc animals scored. None of these integrants mapped to the X chromosome.