Worm Breeder's Gazette 12(2): 32 (January 1, 1992)
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.
Estimates based on genetic and PCR screens for Tc1 insertion suggest that in "high hopper" strains the frequency of a new Tc1 insertion is in the order of 1 x10 +E4per 5 kbp. There can be hotter and colder regions, but on the whole there seem very few exceptionally cold genes. Based on these estimates we think it is possible to freeze away a collection of lines of a high hopper strain, in which Tc1 knock out alleles of most genes are represented. They can be identified by analysis of their DNA. The ultimate goal of this approach would be to have a collection of mutants of all Caenorhabditis elegans genes, making the first step in "reverse genetics", the assessment of the phenotype of a knockout, trivial. In addition the mutants may be used to create more subtle alterations of the genome, by transgene instructed Double Strand Break repair following Tc1 excision (see last WBG).
We have started to set up such a collection. Our strategy is as follows: cultures of 1-4 TR679 worms are started in 24 well plates. Starved F3 larvae are washed off and transferred to 96 well microtiter plates. After sedimentation the supernatant buffer is removed, and from the remaining 50µI worm suspension two 12µl aliquots are taken out. These animals are frozen in 100µl glycerol/buffer, and form the mutant collection in duplicate. With some freezer space, many thousands of cultures can be stored.
To analyze the DNA we do the following: 100µl of lysis solution is added to each of the 25 µl worm pellets in the microtiter plate. The plate is sealed and left at 65°C for a while. Then the lysates are pooled in a matrix: 100 x 96 preps are reduced to 40 pools of each a complexity of 1000. This is done by first combining 20µl from each lysate in a row of a plate, and similarly 20µl from each column of a plate, and by further combination of these in an ordered pattern. This can be visualized by thinking of 10 blocks of apartment buildings of each 10 floors with 96 rooms. Each original prep is represented in 4 of these pools, and the original "address" is represented by the 4 pools in which it is represented (which block, floor, row, and column). The more laborious part of the DNA isolation (phenol extractions, etc.) is only done on these 40 pools; the 1 x10 +E4original lysates are frozen in their microtiter plates. 1 x10 +E4cultures (each started with 1-4 Worms) will represent a complexity of 8 x10 +E4parental chromosomes at max; therefore the collection will not be large enough initially to contain P0 mutations in all genes, but it is a start. We can build more streets later on.
To investigate the presence of a Tc1 insertion in a given gene we do a PCR on the 40 pools with one Tc1 primer and one gene specific primer (see Alice Rushforth and Phil Anderson, WBG 11,5). The sensitivity of PCR is good enough for this complexity. When a positive signal is seen we go back to the original DNA prep, confirm the presence of an insertion, and determine the precise insertion site. The bank can be screened for insertions in genes of interest, but we will also take the global approach: primers that have been used to sequence the genome can be used to screen for insertions in their vicinity. The ideal picture is that we will be able to keep up with the sequencing effort, adding references to cultures with Tc1 insertions to the sequence in acedb. One of the advantages of the approach is that the DNA is available also for other detection techniques (Inverted PCR, vectorette ligation), and some of these techniques might allow us to hand out filters or DNA for others to screen. We are still brooding over this.
Much of this is still a plan. We tested culturing (on agarose to prevent burrowing), freezing and thawing, lysis in plates, pooling, PCR detection, and we started the collection; it looks as if it is doable. We bring it up in the WBG at this time because we would appreciate ideas from others at an early stage.