I tried to transform Physcomitrella patens with Em-GUS cassette by using PEG. After two round selection with 15µg/ml final concentration of hygromycin, I had few transformed lines. However, When I checked the cassette in those transformed lines by PCR with specific primers I did not see those lines have the cassette. I noticed some cells in the moss filaments died after first and second round selections with hygromycin and remained green cells in the same filament started branching when introduced to BCDA medium. Can I consider those survived cells as transforments or is it necessary to whole filament remain green in hydromycin? How can those lines resistant to hygromycin without having the cassette? Can someone explain me this? or any other suggestions to increase protoplast transformation?
Thanks in advance.
Dear Kumudu,
it is hard to find the ultimate cause, there might be many points which maybe could be improved. Amount, purity and preparation of DNA used for transformation, sequence of the construct used, regeneration and proper selection of protoplasts and filaments.
Maybe our latest book chapter on this (Decker, E.L., G. Wiedemann, R. Reski (2015): Gene targeting for precision glyco-engineering: production of biopharmaceuticals devoid of plant-typical glycosylation in moss bioreactors. Methods in Molecular Biology 1321, 213-224) might help with some of the most common questions.
Concerning selection and regeneration: did you follow the common cycles of selection and release in two weeks alterations? Always keep in mind that hygromycin it is not very stable. Take care only to add the hygromycin once the medium's temperature is below 40°C and use freshly prepared plates. If the stock is not freshly opened, sometimes 15 µg/ml does not work, but increasing the concentration to 25 µg/ml works fine. If there are clones surviving the selection, especially if only very few green cells remain in the filaments, I guess that either the hygromycin concentration was not high enough (did you include an untransformed control for the selection?) or the integration of the transgene was not stably into the genome. One main cause for this could be unsufficient linearization of the vector prior to transformation.
Hope this helps to get an idea at which points you could improve the procedure!
Gertrud
Dear Gertrud,
Thank you so much for your explanation. First, I introduced regenerating protoplast on to hygromycin plates after five days, and then I transferred it on to BCD medium after a week. After two weeks on BCD mdeium, then again I transferred it to hygromycin for a week. Again, it was transferred to BCD medium for two weeks. However, I had very slowly growing filaments even after two weeks alternation. I think my hygromycin is not effective to kill the clones.
Kumudu
Dear Kumudu,
our standard procedure is a bit different: we transfer the protoplasts to solid medium only after 10 days. First we grow them for 3 days on plates without antibiotics, than transfer the cellophane sheets to the selective medium and incubate for 2 weeks. In our hands selection only works reliable, if each round of selection lasts 2 weeks. Usually we do 2 cycles of selection and pick the clones from the cellophane for the second selection. If this is not possible (filaments still too small) sometimes a third cycle is useful.
Gertrud
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