@article{3346556,
    title = "Nucleoside Transport and Nucleobase Uptake Null Mutants in Leishmania mexicana for the Routine Expression and Characterization of Purine and Pyrimidine Transporters",
    author = "Aldfer, M.M. and AlSiari, T.A. and Elati, H.A.A. and Natto, M.J. and Alfayez, I.A. and Campagnaro, G.D. and Sani, B. and Burchmore, R.J.S. and Diallinas, G. and De Koning, H.P.",
    journal = "International Journal of Molecular Sciences",
    year = "2022",
    volume = "23",
    number = "15",
    publisher = "MDPI",
    issn = "1422-0067",
    doi = "10.3390/ijms23158139",
    keywords = "adenosine h 3;  Aspergillus nidulans FurD transporter;  blasticidin;  cytidine h 3;  equilibrative nucleoside transporter;  fluorouracil;  formycin B;  guanosine;  guide RNA;  hypoxanthine;  hypoxanthine h 3;  inosine;  LmexNT3 purine nucleobase transporter;  nucleobase transporter;  nucleoside transporter;  purine nucleotide;  puromycin;  pyrimidine nucleoside;  radioisotope;  restriction endonuclease;  RNA polymerase;  thymidine h 3;  Trichomonas vaginalis ENT3 transporter;  Trypanosoma cruzi NB2 transporter;  tubercidin;  unclassified drug;  uracil;  uracil h 3;  uridine h 3, amastigote;  Article;  Aspergillus nidulans;  cell growth;  chromosome 15;  controlled study;  CRISPR-CAS9 system;  expression vector;  gene deletion;  gene expression system;  genetic strain;  genetic transfection;  growth curve;  growth rate;  heterologous expression;  in vitro study;  knockout gene;  Leishmania mexicana;  Leishmania mexicana NT1.1 cell line;  Leishmania mexicana NT1.2 cell line;  Leishmania mexicana NT2 cell line;  Leishmania mexicana strain;  molecular cloning;  nonhuman;  nucleoside transport;  polymerase chain reaction;  prokaryotic cell;  promastigote;  protein expression;  radiochemistry;  radiolabeling;  real time polymerase chain reaction;  resazurin assay;  resistant cell line;  scanning electron microscopy;  Streptococcus pyogenes;  SUPKO cell line;  transport assay;  Trichomonas vaginalis;  Trypanosoma brucei",
    abstract = "The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from Leishmania mexicana (ΔNT1.1/1.2/2 (SUPKO)). SUPKO grew at the same rate as the parental strain and displayed no apparent deficiencies, owing to the cells’ ability to synthesize pyrimidines, and the expression of the LmexNT3 purine nucleobase transporter. Nucleoside transport was barely measurable in SUPKO, but reintroduction of L. mexicana NT1.1, NT1.2, and NT2 restored uptake. Thus, SUPKO provides an ideal null background for the expression and characterization of single ENT transporter genes in isolation. Similarly, an LmexNT3-KO strain provides a null background for transport of purine nucleobases and was used for the functional characterization of T. cruzi NB2, which was determined to be adenine-specific. A 5-fluorouracil-resistant strain (Lmex5FURes) displayed null transport for uracil and 5FU, and was used to express the Aspergillus nidulans uracil transporter FurD. © 2022 by the authors."
}