Worm Breeder's Gazette 14(1): 23 (October 1, 1995)
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.
Laboratory of Molecular Biology, National Institutes of Health/NIDDK, Bethesda, MD 20892-0510.
Inhibition of gene activity using antisense RNA has been a useful tool in the determination of gene function in both prokaryotic and eukaryotic systems. In C. elegans, antisense RNA mediated gene inhibition has been achieved by introducing a promoter driven expression construct of a target DNA fragment in reverse orientation (e.g. unc-22; Fire et al., Development, 1991, 113: 503-514) and by direct injection of the antisense RNA into the hermaphrodite gonad (e.g. par-1; Guo and Kemphues, Cell, 1995, 81: 611-620). In the latter case, injection of antisense RNA to the maternal effect gene par-1 phenocopied a mutation in par-1. We sought to determine if this technique could be utilized in the study of zygotically expressed genes that act later in development. CeMyoD is a member of the family of myogenic basic helix-loop-helix proteins and is involved in the development of body wall muscle (Krause, Bioessays, 1995, 17: 219-228). CeMyoD is stabily expressed beginning at the ~100 cell stage of embryogenesis. We attempted to phenocopy a null mutation in the gene encoding CeMyoD (cc450; Chen et al., Development, 1994, 120: 1631-1641) by injecting antisense RNA transcribed in vitro from the CeMyoD cDNA using T7 polymerase. The antisense RNA reaction (1.0 mg/ml RNA) was injected into each arm of the hermaphrodite gonad at the L4/early adult stage using standard transformation techniques (Mello et al., EMBO J., 1991, 10: 3959-3970). We placed 5-10 injected worms on seeded NGM plates to recover at 20 C for 1-2 days. In the progeny of injected worms, we observed animals that were phenotypically similar to cc450 as seen by their Lumpy/Dumpy appearance and failure to elongate beyond the 2-fold stage. We also observed three other classes of defects: (1) embryos that had more severe defects in morphogenesis than cc450, (2) embryos that elongated beyond 2-fold that were Lumpy, and (3) those that gave rise to larvae with minor head defects. We are not certain what is the cause of this "allelic series", but it may be a dose effect due to the instability or improper partitioning of the antisense RNA or to the interference with processes which utilize other bHLH proteins. On each plate, ~15% of the progeny exhibited a phenotype in the series of defects. No phenocopy defects were seen in progeny of worms injected with a reaction that contained no cDNA template. We have injected the sense RNA and obtain the same results as the antisense RNA injections; the frequency of the phenocopy is similar to that using the antisense RNA. This sense strand effect is similar to what is seen in the phenocopy of par-1 as well as the experience of others using antisense RNA to phenocopy maternal gene products. We have also attempted to phenocopy using antisense oligos. We saw no effect using single antisense oligos (~0.2 mg/ml) or a mixture of several antisense oligos (mix of three oligos at ~0.06 mg/ml each) to CeMyoD. We have injected single stranded DNA to determine if the formation of hybrid RNA leads to the block to gene activity. We were unable to phenocopy cc450 defects using antisense or sense CeMyoD single stranded DNA (~2 mg/ml ssDNA). We have used this technique in the study two other genes that are expressed in muscle and neurons. CeMEF-2 appears to be expressed in all muscles (pharyngeal, body wall, vulval, and male tail) and neurons. CeE12/Da is expressed in cells within the head, tail, and along the ventral region of the embryo at the 2-fold stage; this expression corresponds to regions of neural differentiation. We have cloned these genes using molecular techniques but we have not isolated mutants. We injected worms with antisense RNA from each of these genes. We observed defects in the progeny of worms injected with these antisense RNAs; these defects were limited to tissues that express these genes. In each case tested, the defects were gene-specific, that is, each sequence gave a unique pattern of defects. We have been able to phenocopy the null mutation in the zygotically expressed gene encoding CeMyoD using its cDNA. We have also generated defects in two other zygotically expressed genes that we have cloned. This suggests that the antisense RNA injection technique may provide another reverse genetic tool for the study of zygotically expressed genes in C. elegans.