Antigenic variation studied using VSG RNAi
in place of immune selection.

After performing VSG RNAi, cells undergo a cell cycle arrest and eventually die. However, eventually trypanosomes emerge which escape this arrest.  Analysis of these revertant cells shows that most have switched to a new VSG coat.  As different VSGs typically have very different amino acid sequences, the switched trypanosomes can escape the VSG RNAi operating against the old variant.

Induction of VSG221 RNAi selects for switches
to new VSG variants 

vsg induction

Above: Western blot analysis of protein lysates from T. brucei where VSG221 RNAi was induced for increasing lengths of time.  The VSG221 expressing population disappears, and is replaced by variants which have switched to different VSGs, including VSG1.8.  BIP is used as a loading control.
Modified from Aitcheson et al. (2005) Mol Micro 57: 16-8-1622.

This allowed us to develop a method using VSG RNAi rather than immune selection to select for VSG switch variants.  This provides a very rapid and efficient means for generation of many hundreds of VSG switch variants entirely in vitro.  Various phenotyping and genotyping screens allow us to categorise the different VSG switch variants according to the mechanism that must have been used to mediate the switch.  We can identify the new VSGs that were activated by sequencing the VSG cDNAs amplified by RT-PCR.  This provides an extremely powerful means for studying VSG switching in a very experimentally amenable fashion. 

Reproducible preferential hierarchy of VSG
activation after selection using VSG RNAi

vsg expressed chart

Above: Characterisation of VSG switch variants generated in different VSG RNAi experiments.  The newly activated VSG was identified by sequencing VSG cDNA.  The percentage of switch variants expressing a given VSG is indicated with bars.  We found a reproducible preferential hierarchy in which VSGs were activated.
From Aitcheson et al. (2005) Mol Micro 57: 16-8-1622.

This experimental procedure allows the generation of large numbers of individual VSG switch variants completely in vitro. We would like to use this experimental method to investigate VSG switching in a much more high-throughput fashion than has previously been possible using trypanosome infections in experimental animals. Although there is a clear preferential hierarchy in which VSGs are activated, there is anecdotal evidence that this hierarchy can be periodically at least partially reset. Which epigenetic modifications help maintain a particular hierarchy of VSG expression site activation? How frequently is this reset? Which attributes of a particular VSG expression site affect the preferred switching mechanism that is used to switch the active VSG?