Construction of Artificial Chromosomes for Trypanosome cruzi

MARIANO J. LEVIN; MARÍA A. CURTO; IGNACIO E. SCHOR; HERNÁN A. LORENZI

Seattle, Washington

Simposio; TriTryp Genomes Meeting; 2004

Seattle Biomedical Research Institute

We developed several types of T. cruzi Artificial Chromosomes (TACs). First, we constructed pTAC1 using pBluescript SK+ as a backbone, inserting two head-to-head telomeres, separated by a spacer DNA segmant, and the neo gene. The linear form was stably maintained in T. cruzi cells. Therafter, we generated the pTAC2 vector inserting the pac gene (pac) into pTAC1 in opposite orientation with respect to neo. These TACs were stably maintained in T. cruzi in absence of selective pressure. Next, we constructed the pTAC-odc vector which harbored the odc gene from Crithidia fasciculata allowing T. cruzi to grow in absence of polyamines. pTAC-odc transfectants could grow in the presence of G418 and puromycin, suggesting that genes in TACs are transcribed in both directions, and could survive in SDM-79 medium (polyamine -), showing that TACs can complement T. cruzi auxotrophies. Finally we built pTACgfp in which the odc gene was replaced by a modified version of GFP with an internal EcoRI cloning site. Large EcoRI inserts from T. brucei and C. fasciculata were inserted to pTACgfp. The transfectants are currently under selection.