Worm Breeder's Gazette 16(4): 20 (October 1, 2000)
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.
|1||Dept. MCDB and Neuroscience Research Institute, UC Santa Barbara, Santa Barbara, CA 93106|
|2||Molecular Biology, UT Southwestern Medical Center, Dallas, TX 75390-9148|
Reporter transgenes are frequently used to obtain a preliminary idea of the expression pattern of an endogenous gene and to assess the in vivo function of cis-acting control elements. The most widely used reporter plasmids, made available by the Fire lab, encode the S65C form of GFP coupled with the 3'UTR of the unc-54 gene. We have been studying the expression of med-1,2 and are now able to make some generalizations that might be useful for the study of genes with early zygotic expression.
The med-1,2 genes are direct targets of maternal SKN-1, and are required for SKN-1-dependent specification of EMS cell fates. Global RT-PCR/Southern analysis on individual embryos has shown that med-1 message accumulates in 4-cell (EMS) stage embryos. Our first reporter transgenes, however, showed later expression onset than might be expected from these data. Reporters were constructed by cloning DNA upstream of the translation start sites of med-1 or med-2. The GFP transgenes showed onset of expression in E and MS, just as they are dividing to form MSa/p and Ea/p. Somewhat inexplicably, all med transgenes made to date with lacZ, either alone or fused to GFP, express very weakly or not at all.
We next found that translational fusion 'tag' reporters containing the med-1 3'UTR show expression onset earlier than similar constructs with the unc-54 3'UTR. Such GFP-tagged med-1 transgenes show GFP fluorescence at the birth of E and MS. With a Chemicon anti-GFP antibody, transgene product first appears on the chromosomes of EMS at anaphase. We replaced the GFP coding region of the med-1::GFP::MED-1 reporter with the 35 carboxyl-terminal amino acids of c-MYC, which contains the MYC epitope EQKLISEEDL. Using Boehringer mAb 9E10, we have seen nuclear MYC::MED-1 in late prophase of EMS at the 6-cell stage.
Similar results were obtained with the end-3 gene, which along with end-1 is required for E specification. Both end-3::GFP and end-3::lacZ fusions begin expressing in the E daughters, Ea and Ep, while expression of an end-3::END-3::MYC transgene containing the end-3 3'UTR can be seen in the nucleus and cytoplasm of E.
We conclude that (1) expression of some reporters can occur earlier when their own 3'UTR, rather than that of unc-54, is used, and (2) an epitope tag, such as MYC, may allow even earlier detection of transgene product.