Worm Breeder's Gazette 8(1): 50

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.

DNA Transformation of C. elegans

D. Stinchcomb, J. Shaw, R. Jefferson, S. Carr, W.B. Wood, D. Hirsh

Using techniques developed by Judith Kimble (1), we have been 
microinjecting supercoiled plasmid DNA into the gonad distal arm of 
young adult hermaphrodites.  To detect the inheritance of exogenous 
DNA, progeny of the injected worm are grown for approximately three 
generations and then harvested.  Their DNA is extracted and tested for 
the presence of the injected sequences by hybridization to the plasmid 
DNA.  To date 11 of 119 injections have shown evidence for the 
inheritance of foreign DNA.
The organization of foreign DNA in worm transformants has been 
examined by restriction analysis.  The exogenous DNA is present as 
high molecular weight DNA at high copy number (20-100 copies per 
transformed worm) and arranged predominantly as a head-to-tail tandem 
array.  In one transformant injected with YRp17-Cell0, a plasmid 
containing sequences with worm homology, the exogenous DNA appears to 
have integrated into the worm genome by homologous recombination.
Two independent transformants containing DNA with no extensive 
homology to the worm have been tested for the stability of foreign 
sequences during propagation.  The exogenous DNA is lost in these 
transformants at a fairly high rate.  For a majority of subclones of a 
transformant, only 50% of the progeny in the next generation still 
contain foreign DNA.  By restriction analysis, the foreign DNA in all 
the subclones of a single transformant appears to be identical high 
molecular weight concatamers.  Thus, the exogenous DNA appears to 
segregate as a single unit that is not undergoing rearrangements at a 
high frequency.  The high instability of this unit suggests that 
either the foreign DNA imparts a deleterious phenotype to the worms, 
or the concatamer is extrachromosomal and is being lost during mitosis 
or meiosis.  We are presently investigating these alternatives.  
Recently several subclones have shown high stabilities during 
propagation.  We are now trying to determine the cause of this 
increase in stability.
To assess the frequency of homologous recombination of injected 
plasmid sequences into the worm genome, we have injected both 
supercoiled and linear plasmids containing portions of the unc-54 gene.
Homologous integration of these DNA sequences should result in the 
production of an unc-54 mutation by disruption of the endogenous gene.
We have yet to demonstrate that exogenous DNA can be expressed.  
Plasmids containing the wild-type unc-54 gene or the amber-suppressing 
sup-7 allele have been injected to assess complementation of unc-54 
mutants or suppression of amber mutants at several loci.  In addition, 
we are constructing hybrid genes that could provide a selection or a 
visible screen for transformed worms.  We have recently injected a  -
glucuronidase negative worm with a vector carrying the E.  coli  -
glucuronidase gene fused to the col-1 5'- and 3'- sequences.  
Transformants containing this vector are being tested for  -
glucuronidase activity.  Transformants containing a similar vector 
carrying the bacterial structural gene for neomycin phosphotransferase 
are being tested for resistance to the drug G418.
Dr.  Hans-Borje Jansson, Univ.  of Lund, Sweden joined my program in 
July, 1983.  He is supported by a Swedish National Science Foundation 
Fellowship and a Fulbirght Award.  Concurrently Dr.  A.  Jeyaprakash 
of India joined us.  We are working on trying to characterize surface 
carbohydrates on C.  elegans and attempting to intervene in 
chemotactic behavior using various experimental manipulations.
Bert M.  Zuckerman,