| Summary: | A cDNA microarray was developed with 12 288 elements, consisting mainly of <i>T. gondii</i> tachyzoite (acute infection stage) expressed sequence tags (ESTs). Prior to expression analysis, the fabricated microarray was characterized by utilizing redundancy within the EST set. The redundancy facilitated the evaluation of cDNA hybridization specificity amongst sequences of both low and high similarity. Employing a representative member of a large related gene family as a specific target, optimized experimental hybridization conditions and data filtering methods were developed to investigate the feasibility of employing our highly redundant cDNA microarray to identify potential novel gene family members and/or new gene homologues. The validated cDNA microarray was therewith used to profile tachyzoite gene expression, comparing virulent and avirulent, RH strain and ME49 strain throughout an <i>in vitro</i> infection of primary human fibroblast cells. cDNA microarray hybridization, bioinformatic analysis and a gene filtering procedure based upon statistically significant expression change during infection, revealed ESTs similarly regulated between the two strains. These ESTs represent common components of eukaryotic cellular machinery and involve processes such as cell replication, transcriptional regulation, and protein degradation and turnover. Moreover, 381 ESTs displayed changing expression patterns over the infection course and included potential host immune modulators and genes involved in host cell egression. In contrast, a further 547 ESTs displayed expression profiles differentially regulated between the strains including the parasite specific rhoptry and surface antigens, genes which may ultimately be implicated in intracellular virulence. This work has produced a well characterised, quality controlled <i>T. gondii</i> cDNA microarray, and illustrated its application in studying gene expression regulation <i>in vitro</i>.
|
|---|
