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.
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 properly. 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 chromosome. 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 tetraploids).