Blasticidin antibiotic

Cells were transfected with purified DNA plasmids with the Lipofectamine™ 2000 Transfection Reagent (Invitrogen) according to the manufacturer protocol. 24 h after transfection, Blasticidin antibiotic (10 μg/ml, InvivoGen) was added to the cells for selection. Following selection, the stable ectopic expression of miRNAs was repeatedly assessed using qRT-PCR.

The PfeIF2β disruption plasmid (pCAM-PfeIF2β) was generated by the insertion of a PCR product corresponding to a 5′ portion from the PfeIF2β sequence (846 bp) into the pCAM-BSD vector which contains a cassette conferring resistance to blasticidin. The insert was obtained using 3D7 genomic DNA as template and the primers p18–p19 (with PstI and BamHI sites respectively, Table S1). Attempts to check the accessibility of the PfeIF2β locus were performed by transfecting wild 3D7 parasites with 3′ tagging constructs. To this end, the 3′ end of the PfeIF2β sequence (845 bp, omitting the stop codon) was amplified by PCR using 3D7 genomic DNA and the primers p16–p17 (containing PstI and BamHI restriction sites respectively, Table S1). The 3′ tagging plasmids were generated by inserting the PCR product into the pCAM-BSD-hemagglutinin (HA). Ring stage 3D7 parasites were transfected with 100 μg of plasmid DNA by electroporation, according to Sidhu et al. (2005 (link)). To select transformed parasites, 48 h after transfection, blasticidin antibiotic (Invivogen) was added to a final concentration 2.5 μg/ml. Resistant parasites appeared after 4–6 weeks and were maintained under drug selection.

TTFs were seeded and transduced with pWZL-blast-Klf1 or pWZL-blast-Myb as described under iEP Generation. 48 hr after transduction, cells were passaged and cultured in FEX supplemented with 4 μg/ml blasticidin antibiotic (InvivoGen). The whole medium was replaced with fresh antibiotic-containing medium every 3 days. When a population of resistant TTFs was obtained, cells were seeded accordingly and transduced with pMX vectors to generate iEPs. Blasticidin was added to the medium during reprogramming.