Integration of the VSG2 transgenes into the VSG6 bloodstream expression site (BES) and consequent effect on growth of transgene containing cell lines.
A. Site of integration of transgenes. Electroporation of Acc65I and SacI digested targeting construct (p3952; Supp. Fig. 1) containing either the wild-type or a mutant VSG2 ORF into bloodstream form trypanosomes expressing VSG6 and selection with blasticidin resulted in stable cell lines expressing either wild-type or mutant VSG2 in addition to wild-type VSG6. Sequence of the BES3 containing VSG6 is from Genbank FM162569; intact ORFs are indicated and the vertical ticks are spaced at 10 kbp.
B. Map of VSG2 showing the location of PTCs, at codons 64, 167, 262, 392, 426 and 460. The signal peptide, the N-terminal domain, linkers (1 and 2), the C-terminal domain, and the GPI-anchor signal sequence, and the two N-linked oligosaccharides (NLO and residue number) are shown. The approximate molecular weights (kDa) of wild-type or each of the PTC-containing VSG monomers are shown.