Worm Breeder's Gazette 8(3): 94

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.

Genetic Transformation After Injection of Amber Mutants With the Cloned sup-7 gene

P. Okkema, J. Kimble

We are attempting to develop a transformation system in C.  elegans 
using the cloned sup-7 gene.  The sup-7 gene was chosen since its 
expression will provide a selection of transformed animals among the 
progeny of injected animals.
In our first experiments, animals homozygous for e1107, an amber 
mutation in tra-3, were microinjected with pJK2 (a 9.8kb plasmid 
constructed from the original plasmid provided by R.  Waterston).  
Self progeny of homozygous e1107 animals produce only pseudomales.  Of 
24 animals injected, 2 produced suppressed progeny.  Suppression was 
scored by looking for a perfect hermaphrodite tail under the 
dissecting scope and confirming this with Normarski.  Controls 
performed during tRNA injections (Kimble et al., 1983) showed that 
e1107 pseudomales never have either perfect hermaphrodite tails or 
perfect hermaphrodite gonads.  Among the transformed progeny of one 
injected animal, 3 had normal shaped hermaphrodite gonads producing 
only sperm and 6 had intersexual gonads producing only sperm and 6 had 
intersexual gonads producing only sperm. [repeat in original]  Thus, 
although suppressed, the animals were sterile.
We next injected pJK2 into animals carrying e364, an amber allele of 
dpy-13.  The injected strain also carried e678 so that suppression of 
the Dpy phenotype might generate Lon animals to facilitate the screen 
for suppression.  Uninjected e364 e678 animals produced 0/105 Lon 
animals in a controlled screen.  After injection, no Lon worms were 
found in F1 or F2 progeny, but Lon worms were found in the F3.  In one 
injection, Lon animals were seen through F6, but F7 and F8 progeny 
were all Dpy.  Lon animals appeared sick, but produced small broods 
containing mostly Dpy and a few Lon worms.  Dot blots of DNA isolated 
from the F1-F3 progeny of the injected worms showed that a yeast 
sequence present on the plasmid as a molecular marker was also present 
in each of the populations with Lon worms.  (However, this sequence 
was also found in some populations with no Lon worms.) Uncut DNA 
isolated from populations with Lons was used to transform bacteria 
selecting for ampicillin resistance.  These bacterial transformants 
contained the same plasmid that was injected, as judged by restriction 
These results show that injected DNA is inherited through several 
generations and that the injected sup-7 gene is expressed.  We are 
currently cloning random nematode DNA fragments into a plasmid 
containing the sup-7 gene in an attempt to stabilize the plasmid and 
thereby reduce the deleterious effects of sup-7 expression.