Differentiation of Trypomastigotes into Extracellular Amastigotes
1:54
Electroporation of Extracellular Amastigotes
3:01
Monitoring the Growth of Knockout Cells
4:35
Results: Growth Monitoring and Phenotypic Differences of Knockout Parasites
5:41
Conclusion
Transcription
It is difficult to use the host infection stage of T.cruzi in experiments because amastigote is an obligate intracellular parasite. Our culturing technique allows the amastigote to temporarily replicate outside of the host cell, so we can perform
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Here, we describe a protocol to introduce a gene knockout into the extracellular amastigote of Trypanosoma cruzi, using the CRISPR/Cas9 system. The growth phenotype can be followed up either by cell counting of axenic amastigote culture or by proliferation of intracellular amastigotes after host cell invasion.