we have had some user requests where it would have been nice to have the genome sequence of the Sf9 or Sf21 cell lines available. E.g. we were trying to co-express tRNA from Drosophila in Sf21 cell using baculovirus, but the tRNA was not detectable by Northern blot. It would be unusual that tRNA promoters and other motifs triggering the transcription of endogenous Sf tRNA are so different that Drosophila tRNA motifs would not work (Drosophila tRNAs were used in yeast, so there seems to be a high level of conservation).
The question is, does anyone know of a fully sequenced Sf genome or a project working on that except the Spodobase (http://bioweb.ensam.inra.fr/spodobase/) which seems to be stuck at a very early stage and is far from being useful?
Would anyone else be interested in the genome sequence? I have read that these cell lines show a high tendency to be polyploid. I'm not an expert in that field, but I don't think that it would be a problem if there is more than one copy of a chromosome as long as Í know the sequence of a particular gene or promoter. We have the sequencing machines available in our institute, so the only question is whether we should do it or not.
Let me know what you think about this or if you have any suggestion, e.g. using the Sf larvae instead of one cell line?
Very co-incidental, I had a meeting with a colleague 5 min ago, who was interested in the exact same thing and I logged on to post the question, but you beat me to it ! In brief, they are working with drosophila proteins and are interested in doing a pull-down using proteins expressed in Sf9 cells under the assumption that interacting proteins are conserved in Spodoptera. However, mass-spec have difficulties identifying the proteins as the Sf protein database is incomplete. Hence, for such purposes knowledge of the Sf proteome would be very valuable, so I strongly support your suggestion.
I would think that by using a native source, such as the larvae, one would avoid complications with cell-lines having elimated certains parts of the genome ?
I'm happy that I'm not the only one who is in favour of such a sequencing project. I agree that the decision about what exactly should be sequenced is the most important issue. Ideally one should sequence both the average genome of the organism and the most widely used cell line(s) although there might be significant differences in the individual cell lines (which one would you use as the reference cell line?). It's just a matter of costs.
If I would have to do it on my own budget my priority would of course be the main cell line that we are using whatever differences there might be with any other cell line or the larvae genome. In case there would be some support by other groups sharing the costs (~1500€ per sequencing run, i.e. per genome), we could go for a deeper look at the differences, especially for the goal of a comprehensive mass spec database this approach would be better.
I just try to use this way of communication to anwer the questions. The genome of Sf9 are not very stable (Ref.Biotech Prog 2002, 18, p623-8), have many small chromosomes and are suffering from polypoidy 2n-4n. The initial annotation of ESTs is described in Landais et al. (Bioinformatics 2003 Vol 19, no18, p 2343-50). The sequence annotation will take a while but afterwards its is much eacher to compare the changes you made, or happened to your cell line. Indeed you could come up with a comparison like the one done for CHO. The strain engineering will be much more focussed if the bioinformatic data of the genomes are available.
The HZI can use its Genome Analysis Platform for this purpose. I actually already asked them one month ago. Does anyone know the total genome size of a 2n diploid genome of Sf9?
Many want to join in such a project. We should start with one to max three cell lines (one of each Sf9, Sf21 and Hi5) and then decide depending on the success of the project (including annotation) how we could proceed further. I will definitely want to sort out the best location for our integrating cassettes in these genomes. I will ask about the annotation of the genome within our institute
Best regards Joop