Worm Breeder's Gazette 10(2): 91

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.

A Few Notes on Transformation Technology

Andrew Fire

How Injections Work: I am now almost exclusively using injection 
into oocytes of young adults to assay transient expression and make 
transformed lines.  In order to do this easily it is crucial that the 
worms be in a fairly narrow window of desiccation on the agarose pads: 
immobile and dry enough to be easily injected but damp enough so that 
the oocyte nuclei can be seen under nomarski 400x.  I think that under 
optimal conditions the insides worm actually turn into a gel, which 
allows very facile and directed injection.  Enhanced gelation at low 
temperature is presumably why injection is so much easier in a cool 
room.  Since my lab hovers around 22 C, I have modified the procedure 
somewhat in that I grow the animals to be injected on the wettest 
plates that can be found, and use a fairly wet plate (~1-2 days old) 
as an intermediate between the growth plate and the injection pad.  
When possible I cool the animals down above an ice bucket (~8 C) for 
15-30 min before injection.  In order to keep them from drying out on 
the pad it is advantageous to find proper spacing between animals.  
The animals are becoming dehydrated as their water diffuses into the 
agarose pad underneath.  If the animals are placed close together, 
then the zones of water diffusing out from different worms will 
overlap, which will slow down the desiccation.  If they are placed too 
close together then not enough desiccation occurs and they are 
difficult to inject.  Play with pad thickness, intermediate plate 
wetness, time between mounting and injection, and animal spacing.  
Since recovery after injection is presumably the reversal of the 
gelled state, one might expect this to proceed much more readily at 
higher temperature.  This is indeed true; animals seem to recover much 
more efficiently at 25 C, and this is now the standard (usually 2-7hrs 
of recovery).
On DNA Quality: Our DNAs are no longer prepared by running CsCl 
gradients.  Instead, we have been using a cleaned up plasmid miniprep 
procedure.  The DNA obtained from CsCl gradients of large scale preps 
is actually less clean than after the cleaned up minipreps.  Briefly, 
plasmids in E.  Coli DH5alpha are grown up overnight in 17mm culture 
tubes with 3ml 2xYT medium.  1.5 ml of this is subject to the standard 
alkaline/SDS miniprep procedure and cleared by a LiCl precipitation 
step.  This material is then treated with RNAse, then Protease K, 
extracted with Phenol/ChCl3 then CHCl3, precipitated twice with 
ethanol and resuspended in standard TE (detailed protocol on request). 
DNA prepared just through the lithium stage is injectable, but there 
is still a considerable amount of protein and RNA at this stage, and 
variability in cleanliness between different crude minipreps tends to 
cast some doubt on negative results.  In general all of our important 
negative results are repeated with two different minipreps or if 
possible with two different plasmid isolates.
Our current DNA concentrations are 60-150 g/ml.  There still doesn't 
seem to be any obvious dependence of copy number or transformation 
frequency on DNA concentration.  If a plasmid seems to be having 
trouble giving heritable transformants, I usually drop the DNA 
concentration 2 fold.
Types of Transformation: First some words.  A transformation event 
is an effect mediated by injected DNA.  In transient transformation 
the animal which is transformed does not pass the trait on to its 
progeny.  In heritable transformation the transforming trait is passed 
on to the progeny of the original transformed animal.  In non-
chromosomal transformation the transforming DNA is present as a 
separate molecule and not associated with a C.  elegans chromosome or 
a large piece thereof.  Chromosomal transformation consists of joining 
of injected DNA sequences with C.  elegans chromosomes.  A subset of 
this, integrative transformation, consists of the integration of 
transforming DNA into or onto fully functional C.  elegans chromosomes.
Homozygote transformants are transformed lines that grow as 
uniformly transformed stocks.  The term 'stable transformant' seems to 
mean something different to everybody and it is hereby retired.
Given these words the take home lesson to date is that almost 
anything can happen.  In selection schemes, such as most sup-7 vectors,
that yield low copy number transformants, the injected DNAs in 
transformed lines are either present as integrated copies, which 
should behave in some sort of mendelian fashion, or as a few copies 
attached to a piece of a normal C.  elegans chromosome which has 
broken off (an injection induced 'free dup').  These free dups are 
visible with DAPI stain and usually contain much more DNA than can be 
accounted for by the number of copies of the transforming DNA (D.  
Albertson p.c.  and our unpublished observations).  Generally about 
half of the low copy number transformants are these hybrid dups.  
Their genetic behavior can be bizarre with highly variable 
transmission ratios and occasional odd phenotypes, but this rarely 
effects the ability of genes on the transforming molecules to function 
Transformation selections that can be satisfied by high copy number 
non-chromosomal arrays of the injected DNA will generally yield these 
long arrays as the primary product.  This is particularly true with 
cytoplasmic as opposed to nuclear injection (nuclear injection is 
apparently required for chromosomal transformation; maybe the 
chromosomes have to be broken by the needle).  In several cases the 
high copy number tandem arrays have actually integrated into a 
At one point it was believed that the large extrachromosomal tandem 
arrays were not properly expressed in terms of tissue specificity.  
This does not appear to be the case with unc-54.  Long tandem arrays 
of the unc-54 gene express the protein at high levels.  The over-
expressed protein accumulates without assembling properly, but is 
limited only to body wall type muscles.  It is worth keeping in mind, 
even if genes present on the tandem arrays can be properly regulated, 
that the presence of 1000 fold excess of a specific gene or product 
within a given cell is relatively likely to have bizarre effects due 
to antisense, over-expression, etc.
Integrative Transformation with Linear DNAs has been tested using 
the unc-54 gene (the formation of large non-chromosomal tandem arrays 
from linear DNA has been demonstrated by Shaw et al.  and A. F. and D. 
Moerman).  Integrative transformants were obtained with linear unc-54 
DNA [PvuII cut punk54] at a frequency comparable to that for circular 
DNA; in general the ends of the DNA seem to be slightly modified, 
since the end restriction enzyme site is lost.  The linear DNAs 
function surprisingly poorly in transient expression, giving only a 
fraction of the signal observed with the corresponding circle.  This 
could be due to instability or inactivity of the free linear molecule, 
or alternatively to efficient concatenation of the injected molecules. 
Linearized sup-7 plasmids are as active a circles for transient 
expression in the distal tip region of the gonad, but much less active 
for transient expression when injected into oocytes.
Homologous Recombination Between Injected Molecules was tested by 
injecting two plasmids containing deletions in different parts of the 
unc-54 gene.  Each of these plasmids by itself is inactive.  A single 
transformed line (an integrant) has been derived from co-injection of 
the two plasmids.  A band corresponding to the recombined fragment can 
be observed on southern blots of the strain (with several enzymes) as 
well as the two parental bands.  Thus there is at least some level of 
recombination between injected molecules.  We are now testing for the 
presence of the reciprocal fragment and other nonselected 
recombination fragments to assess recombination frequency.
There was no transient expression after co-injection of the two 
deletions.  This suggests that the burst of F1 transient expression 
after injection of oocytes with unc-54 constructs might very well be 
due to partitioning and expression of the injected circular DNAs 
rather than formation of large tandem arrays outside the germline.  
Tetraploid transformants and 'transformants': Judith Kimble originally 
discovered that microinjection can induce triploids and tetraploids.  
For some selections this is a concern since polyploid animals can 
appear rescued, and are generally observed frequently enough to be a 
problem.  For selections that polyploidy does not interfere with, it 
turns out that a fraction of the real transformants are tetraploid, or 
more often triploids that eventually yield tetraploid stocks.  The 
polyploids are very long and exhibit some odd segregation, but in 
general can be treated and analyzed as are other transformed lines.
Gene Fusion Vectors and Requirements for Expression: If you're 
designing fusion constructs to use a reporter gene for monitoring gene 
expression keep in mind that the insides of gene are far from inert.  
There was a really good nematode splice signal in B-gal.  I'm not sure 
how this affects expression but the site is taken out in our active 
unc-54: gal fusions.  Likewise please note in the accompanying article 
on unc-54 expression that worm genes may need more than what is on 
their 5' end to make product efficiently.  Different sup-7 vectors 
have different properties: A number of sup-7 vectors constructed by R. 
Waterston and myself have been used as selectable markers for 
transformation.  In general these have been cloned in such a way that 
sequences upstream the sup-7 gene (-20 and beyond) derive from the 
particular site used in the cloning vector.  These plasmids all give 
chromosomal transformants, but the average copy number tends to vary 
between constructs.  The pAST plasmid generally gave 2-8 copies while 
constructs in pAST 19b usually give only one or two copies.  For some 
experiments low copy number is advantageous, but it is worth keeping 
in mind that a circular molecule has to break somewhere and hence 
disrupt some region in order to integrate as a single copy.  [Other 
labs (e.g.  Bloomington, St.  Louis) have further information on copy 
numbers for the different pAST vectors.
Oddly enough the phenotypes of the transformed lines also seem to 
depend on the vector used.  For each the most strongly transformed 
lines are generally sickly.  For pAST these lines are somewhat dumpy, 
whereas with pAST 19b the strongly transformed animals are long (not