Worm Breeder's Gazette 9(1): 85

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More on transgenic worms expressing sup-7 DNA

A. Fire

Amber alleles of the gene tra-3 provide a sensitive assay for amber 
suppression; without suppression, tra-3 animals are intersexual and 
sterile above 20 C.  Expression of suppressor tRNA either in the 
mother or zygote results in animals that are phenotypically 'rescued' 
and hence fertile at 20 C.
The initial goal was to assay for transient expression of DNA 
following microinjection.  Kumble, et. al., (Nature 299, 456) had 
shown that injection of a tRNA fraction from the sup-7 strain into the 
distal gonad arm of tra-3 animals rescued some of the resulting 
progeny.  I obtained similar results upon injection of the sup-7 gene 
contained in a supercoiled plasmid.  The observed rescue resulted 
almost entirely from transient expression since none of the rescued 
progeny expressed sup-7 in their own gonads.  In optimizing transient 
expression, a strong dependence on the site of injection was observed. 
The most efficient transient expression was obtained following 
injection near the rapidly dividing nuclei around the distal tip.  
Some transient expression was also obtained following injection near 
the bend in the gonad arm and into oocytes.  Surprisingly, injection 
into the center bulge of the distal arm, which until now has been 
standard for most studies, yielded little or no expression.
Transient expression should provide a tool useful in analyzing 
expression of genes active during oogenesis and early embryogenesis.  
For most applications, however, one needs transgenic lines expressing 
a DNA segment of interest.  Hence a large number of tra-3 animals were 
injected with sup-7 containing DNA constructs and transformed progeny 
selected by continued growth at 20 C.  To date, ten independent 
transgenic lines have been established; each has continued to breed 
since establishment (ten to forty generations).  The nature of 
injections seem to be critical for stable transformation; the most 
successful sets of injections have been those in which each worm was 
injected multiple times (six to ten) aiming for nuclei around the turn 
in the gonad.  Although the first transformants were from injections 
of sup-7 cloned inside the ends of the transposable element Tc1, it 
has subsequently proved unnecessary to have any Tc1 sequences in cis 
or trans.  Indeed the highest frequencies so far have been observed 
after injection of a 4kb sup-7 plasmid with no transposon elements; 
three transgenic lines were derived from 169 injected parents.  At 
present, doing multiple injections near the gonadal turn, I can inject 
and recover about 80 animals/day.  Including time to follow the 
injected animals and maintain reagents and stocks, about two and one-
half days of full-time work are needed per transformed line.
The existing transformants are being characterized both genetically 
and by Southern blots.  Homozygous strains have been derived for five 
of the ten transformants.  In four cases the suppressor locus has been 
mapped to a chromosome by standard genetics; one suppressor maps on 
chromosome I, one on chromosome III and two on chromosome II.  Of the 
transformants so far analyzed using Southern blots, each appears to 
contain several copies of the injected plasmid; these copies appear to 
have been linearized at different places and joined together.  
Although some rearrangements are observed, it is encouraging that 
large (4-7 Kb) fragments from the initial plasmids have apparently 
been preserved without internal rearrangement.  In making some of the 
transformed strains, the sup-7 containing plasmid had been co-injected 
with a second, unselected plasmid.  In the two such strains so far 
analyzed, sequences from the second plasmid are also present in the 
transformed line.  This may be useful in co-introducing various genes 
of interest.