Worm Breeder's Gazette 10(2): 97

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Transformation to the Rescue!

Ian Hope, Roger Hoskins, Andrew Spence, John Sulston, and Danielle Thierry-Mieg

Figure 1

In order to make good use of the genome map (see update, this issue),
we need an efficient means for searching out loci that have been 
shown genetically to lie within a limited region.  Transformation 
rescue, developed by A.  Fire (EMBO J.  5(10) pp.2673-2680 1986), 
offers a potentially more powerful, and also more stringent, 
identification of loci than has previously been available.  It is, of 
course, also central to the identification of functional elements by 
reverse genetic analysis (eg Fire, WBG 10(1)).
Since hearing from Andy Fire that he had been successful in 
obtaining transformants with DNA minipreps, we have been using a very 
simple protocol to prepare DNA for injection.  Cosmid DNA is extracted 
from 2ml bacterial culture by a standard alkaline lysis procedure, 
ethanol precipitated and redissolved in 200 l TE.  An equal volume of 
4.4M LiCl is added, and the mixture is left on ice for an hour.  RNA 
and other contaminants are pelleted by centrifugation and the DNA is 
precipitated from the supernatant with two volumes of ethanol.  The 
DNA is reprecipitated from 100mM KOAc (Fire,1986) and dissolved in 10 
l of injection solution.  The size of the cosmid DNA should always be 
checked on an agarose gel to guard against large deletions.
We use the injection set-up and procedure of Fire (1986).  We are 
following his advice in injecting under paraffin oil rather than 
Voltalef.  We find that it is unnecessary to have Lucifer Yellow in 
the injection solution as successful injection is easily visible with 
Nomarski optics.  We periodically verify that the needle is flowing by 
touching it to the agarose pad.  It is possible to obtain rescued 
animals by injecting nuclei or by actively avoiding them, but we do 
not know what influence the site of injection has on the transmission 
of the rescuing DNA.  We have observed rescue arising in the offspring 
of unrescued progeny of injected animals.
Whenever possible, when searching for a gene, we inject a set of 
cosmids which overlap to the extent that each region is represented in 
two cosmids.  This should control against the presence of small 
rearrangements in any given cosmid and increase the chance of having 
the entire gene on at least one cosmid.
Our experience is admittedly limited, and some mutants may well 
prove difficult to rescue, but we have not yet encountered a gene 
refractory to transformation or a DNA clone which is known to span a 
gene and yet fails to rescue mutants in that gene.  It is worth 
pointing out that unc-32 and unc-30 are, as far as we know, the first 
cases of nematode genes being cloned on the basis of genetics, 
physical linkage, and rescue, without the involvement of polymorphisms 
in mutants.  As the physical map becomes more crowded, this approach 
to cloning loci can be expected to become increasingly general and 
[See Figure 1]

Figure 1