Worm Breeder's Gazette 15(1): 28 (October 1, 1997)

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.

YAC Rescue Made Easy

Andrew G. Davies, Jocelyn E. Shaw

Dept. of Genetics and Cell Biology University of Minnesota 1445 Gortner Ave. St. Paul, MN 55108 U.S.A.

We would like to report a simple method of YAC preparation for
transformation of C. elegans that avoids isolation of the YAC DNA away
from the yeast genomic DNA.  We have found that injection of total
genomic DNA isolated from a yeast strain bearing the YAC of interest
can produce rescue in 10-50% (about 30% is typical) of stable lines.
The YAC DNA is prepared using a standard yeast genomic DNA isolation
method (spheroplast formation followed by lysis and potassium acetate
protein precipitation).  The DNA is purified by banding in CsCl under
ultracentrifugation.  Other methods of DNA purification (e.g. QIAGEN
genomic DNA columns) also appear to work (C. Spike, unpublished data).
Injection of the yeast/YAC DNA at concentrations between 50-150 ng/ul
(75-100 ng/ul is recommended) along with pRF4 (Mello et al. (1991) EMBO
J. 10:3959-3970) at 100 ng/ul, as a transformation marker, is
sufficient to produce transformed progeny and stable transformed lines
at a useful frequency.
Using this method we have been able to achieve rescue of a maternal
effect lethal gene, gut-4, and the zygotic synthetic lethal combination
mec-8; sym-1 (see Davies, Shaw and Herman, this issue).  Injection of
other YAC-bearing strains known not to cover these genes fail to
rescue, making it unlikely that it is yeast genomic DNA that rescues
the mutant phenotype in these cases.  In addition, two genes known to
span relatively large regions of genomic DNA have been rescued using
this method, lin-29  (J. Bettinger and A. Rougvie, pers. comm.) and
unc-7 (T. Starich and J. Shaw, unpublished data).  The detailed
protocol we have used for preparation of yeast DNA will be available at
the Ambros lab protocol site