We in our lab have been observing massive amount lysis of RBC in trasnsfected cultures of Plasmodium falciparum. This observation has been particularly consistent after 2 cycles of selection pressure (here the selection pressure was with 2ug/mL Blasticidin ). We have never observed anything of this sort with our previous transfections, where we had used Albumax II. The only thing different this time was that we used heat inactivated human serum instead of Albumax. Has anyone observed anything similar to this in their lab?
PS: our wildtype cultures are growing perfectly fine in human serum containing media, infact the growth in human serum containing medium is much faster when compared to Albumax containing media.
What did you transfect the cells with? It's likely that the nucleic acids you're adding are causing adverse effects if the medium is fine. You should also test control samples with your selection marker to make sure you're not using too high of a concentration. You should also transfect a detectable tag (like GFP) to make sure you're conferring a selection marker to the cells. If it turns out that you're having issues with transfection efficiencies, try a lipofection-based approach (like https://altogen.com/product/altofect-transfection-reagent/) using high concentrations of transfection reagent. It is possible that because you are transfecting bacteria, they may exhibit less responsiveness to classic transfection of mammalian cell lines.
What did you transfect the cells with? It's likely that the nucleic acids you're adding are causing adverse effects if the medium is fine. You should also test control samples with your selection marker to make sure you're not using too high of a concentration. You should also transfect a detectable tag (like GFP) to make sure you're conferring a selection marker to the cells. If it turns out that you're having issues with transfection efficiencies, try a lipofection-based approach (like https://altogen.com/product/altofect-transfection-reagent/) using high concentrations of transfection reagent. It is possible that because you are transfecting bacteria, they may exhibit less responsiveness to classic transfection of mammalian cell lines.
Most of our vectors have a GFP-fusion tag. Among all the transfection protocols of Plasmodium falciparum, we used the most widely used method described by Wu et al (1995), it is also known to give the most stable transfection among other known methods. We have always been using this method and it has been working well in our lab. But we faced such a bizarre and explainable problem only this time. Plasmodium falciparum is a protozoan and the transfection methods used for the same , takes care of the necessary modifications of classic electroporation protocols used for mammalian cells .
There are some publications where new methods have been used. In particular the lab in California (DeRisi) and UK (Blackman) improved the electroporation protocol and results a lot. Malar J. 2012 Jan 18;11:22. Plate-based transfection and culturing technique for genetic manipulation of Plasmodium falciparum. Caro F, Miller MG, DeRisi JL + Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle. Authors: Collins CR1, Das S, Wong EH, Andenmatten N, Stallmach R, Hackett F, Herman JP, Müller S, Meissner M, Blackman MJ. In: Mol Microbiol (2013) 88(4): 687-701
There are some publications where new methods have been used. In particular the lab in California (DeRisi) and UK (Blackman) improved the electroporation protocol and results a lot. Malar J. 2012 Jan 18;11:22. Plate-based transfection and culturing technique for genetic manipulation of Plasmodium falciparum. Caro F, Miller MG, DeRisi JL. Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle. Authors: Collins CR1, Das S, Wong EH, Andenmatten N, Stallmach R, Hackett F, Herman JP, Müller S, Meissner M, Blackman MJ. In: Mol Microbiol (2013) 88(4): 687-701
There are some publications where new methods have been used. In particular the lab in California (DeRisi) and UK (Blackman) improved the electroporation protocol and results a lot.
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