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.

Antisense RNA inhibition of zygotic genes expressed in muscles and neurons.

Morgan Park (mp130c@nih.gov), Michael Krause

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
 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
 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
 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.