The chloramphenicol acetyltransferase vector as a tool for stable tagging of Neospora caninum

Autor(es)

Pereira, Luiz Miguel ^{[1, 2]} ; Yatsuda, Ana Patricia ^{[1, 2]}

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, BR-14040903 Ribeirao Preto, SP - Brazil

^[2] Univ Sao Paulo, Nucleo Apoio Pesquisa Prod Nat & Sintet, Ribeirao Preto, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Molecular and Biochemical Parasitology; v. 196, n. 2, p. 75-81, SEP 2014.
• Citações Web of Science: 5

Resumo

Neospora caninum is an obligate intracellular Apicomplexa, a phylum where one of the current methods for functional studies relies on molecular genetic tools. For Toxoplasma gondii, the first method described, in 1993, was based on resistance against chloramphenicol. As in T. gondii, we developed a vector constituted of the chloramphenicol acetyltransferase gene (CAT) flanked by the N. caninum dihydrofolate reductase-thymidylate synthase (DHFR-TS) 5' coding sequence flanking region. Five weeks after transfection and under the selection of chloramphenicol the expression of CAT increased compared to the wild type and the resistance was retained for more than one year. Between the stop codon of CAT and the 3' UTR of DHFR, a Lac-Z gene controlled by the N. caninum tubulin 5' coding sequence flanking region was ligated, resulting in a vector with a reporter gene (Ncdhfr-CAT/NcTub-tetO/Lac-Z). The stability was maintained through an episomal pattern for 14 months when the tachyzoites succumbed, which was an unexpected phenomenon compared to T. gondii. Stable parasites expressing the Lac-Z gene allowed the detection of tachyzoites after invasion by enzymatic reaction (CPRG) and were visualised macro- and microscopically by X-Gal precipitation and fluorescence. This work developed the first vector for stable expression of proteins based on chloramphenicol resistance and controlled exclusively by N. caninum promoters. (C) 2014 Elsevier B.V. All rights reserved. (AU)

• Processo FAPESP: 05/53785-9 - Proteínas envolvidas na invasão de células hospedeiras por protozoários apicomplexas como potenciais componentes para métodos profiláticos
• Beneficiário: Ana Patricia Yatsuda Natsui
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores
• Processo FAPESP: 09/07713-7 - Avaliação funcional da proteína anônima relacionada à trombospondina 2 de Neospora caninum (NcMIC2-like1)
• Beneficiário: Luiz Miguel Pereira
• Modalidade de apoio: Bolsas no Brasil - Doutorado