Worm Breeder's Gazette 10(2): 97
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.
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 convenient. [See Figure 1]