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Development of Leishmania Species Strains with Constitutive Expression of eGFP
JoVE Journal
Genética
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JoVE Journal Genética
Development of Leishmania Species Strains with Constitutive Expression of eGFP

Development of Leishmania Species Strains with Constitutive Expression of eGFP

DOI:

10:03 min

April 21, 2023

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Capítulos

  • 00:04Introduction
  • 00:27Amplification and Insertion of the eGFP Gene into the pLEXSY‐2.1 Vector
  • 02:39Linearization of the pLEXSY Expression Plasmid
  • 03:26Transfection of L. panamensis and L. donovani by Electroporation
  • 05:00Polyclonal Selection in Culture
  • 06:41Confirmation of Genomic Integration
  • 07:09Cloning by Limiting Dilution
  • 08:12Results: Confirmation of eGFP Cassette Expression and Chromosomal Insertion
  • 09:40Conclusion

Summary

Tadução automática

Here, we describe the methodology used for generating L. panamensis and L. donovani strains expressing the gene for eGFP as a stable integrated transgene using the pLEXSY system. Transfected parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity in both species were selected for further use in drug screening assays.

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