Abstract:
: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.
journal_name
Mol Biochem Parasitoljournal_title
Molecular and biochemical parasitologyauthors
Pereira LM,Yatsuda APdoi
10.1016/j.molbiopara.2014.08.001subject
Has Abstractpub_date
2014-09-01 00:00:00pages
75-81issue
2eissn
0166-6851issn
1872-9428pii
S0166-6851(14)00092-9journal_volume
196pub_type
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journal_title:Molecular and biochemical parasitology
pub_type: 杂志文章
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journal_title:Molecular and biochemical parasitology
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