Worm Breeder's Gazette 11(1): 18

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.

Worm Transformation Gets Rolling

C. Mello, V. Ambros, J. Kramer and D. Stinchcomb

We have obtained C.  elegans DNA transformants at a high frequency 
by microinjection.  Our procedure is a simplified version of the 
microinjection techniques devised by J.  Kimble, D.  Stinchcomb, J.  
Shaw, and A.  Fire.  Based on the number of injections required to 
obtain an extrachromosomal transformed line, we estimate the the 
modified method is 10 to 100 times more efficient than previously 
described techniques.
We began injecting a 7 kb plasmid, pRF4, containing the dominant 
right roller gene, rol-6(su1006) to determine its utility as a 
dominant marker for transformation.  We injected 8 to 15 oocytes in 
each of approximately sixty worms and obtained 18 F1 Roller progeny, 
two of which proved to be germline transformants.  In contrast, by 
injecting each syncytial distal gonad arm of 19 hermaphrodites (two 
injections per worm), we obtained 263 F1 Rollers, 10 to 20% of which 
proved to be germline transformants.
Aside form the obvious advantage of doing fewer injections to obtain 
a transformed line, there are several other advantages of syncytial 
injection over oocyte injections.  1.) The target is much bigger so 
even a novice can obtain plenty of transformed worms.  2.) The worms 
suffer less: which a.)improves lab karma, b.) increases worm viability,
and c.) allows one to use larger needle tips (e.g., to inject viscous 
DNA samples).  3.) Worms do not dry out and do not need any special 
recovery.  Injected worms may simply be removed from the pad with 
sterile M9 and placed-directly onto a seeded plate.
To perform syncytial injections, we focus on the grainy textured 
area in the center of the distal gonad arm.  The tip of the needle is 
brought into the same focal plane and the stage is moved, pushing the 
worm into the needle tip.  Once the needle is centered within the 
syncytium, pressure is applied to the needle.  During injection, a 
wave front moves slowly in both directions from the site of injection, 
literally filling the gonad with DNA solution.  It is easier to fill 
the gonad if each arm is injected only once.  Additional holes seem to 
allow the DNA to leak out.  We routinely inject DNA at 10 to 200  g/ml 
in the injection buffer described by A.  Fire.  Transformation 
frequency appears to plateau above 20  /ml.
These injections require a needle that flows copiously.  We have 
found that good needles can be reproducibly obtained by treatment with 
hydrofluoric acid (HF).  A needle is loaded into its carrier and 
pressurized to 50 lbs./sq inch.  The tip of the needle is inserted 
into a small drop of HF on a plastic petri dish.  After approximately 
one second, when the needle starts to bubble, it is transferred to a 
drop of water to rinse the tip.  The procedure is repeated to obtain 
larger tip openings.  We have found that the optimal tip size will 
permit bubbling in the drop of water when pressurized as described 
Cotransformation frequency is high with this technique.  Seventeen 
independent lines were obtained by coinjecting the rol-6 plasmid with 
other plasmids or cosmids.  Hybridization analysis of DNA prepared 
from the transformed lines showed that all (100%) contained both 
coinjected sequences.  We have used coinjection to rescue unc-31(e928) 
with the phage containing the unc-31 gene provided by R.  Hoskins and 
emb-9(cg34) with a phage containing a basement membrane collagen gene.
Coinjecting certain sequences appears to reduce transformation 
frequency.  Worms injected with an equal molar ratio of sup-7am and 
rol-6 plasmid DNA produced fewer rolling F1 worms and never germline 
transformants.  Similar results were obtained with a cosmid in the lin-
4 walk, B0243.  We found that by reducing the ratio of the 'poison' 
sequence to rol-6 plasmid, we were able to obtain cotransformed lines.
The high efficiency of this transformation procedure has led us to 
consider less abundant sources of worm DNA for transformation 
experiments .  In another abstract in this issue (D .  Levitan, et al .
), we describe the rescue of par-2(it5ts) by microinjecting DNA 
isolated from a yeast strain bearing a worm artificial chromosome (YAC)