Generation of VPS8D knockdown strains
To generate VPS8D knockdown strains, a construct was designed as
shown in Fig. 1B, by first PCR amplifying ~500 bp of
genomic VPS8D gene coding sequence with two sets of primers, one
set engineered with ApaI and XhoI restriction sites and another set with
PmeI and SmaI restriction sites. These two fragments were cloned into
PCRII-I3 so that they were inverted relative to one another. The rDNA
backbone vector pIBF (Howard-Till and Yao, 2006) was digested with with
ApaI and PmeI to release the BFP fragment. In parallel, the VPS8Dhairpin cassette was excised from the PCRII-I3 backbone with ApaI and
PmeI, and the ~1kb insert then gel purified and ligated
into the site of the excised BFP fragment in the pIBF backbone.
Approximately 20µg of this plasmid was used per biolistic
transformation. In addition to the rDNA-based VPS8D hairpin
strains, we also engineered a construct to target the VPS8Dhairpin cassette to the macronuclear RPL29 locus. In this case,
theVPS8D hairpin cassette was cloned into pBSICC-rpl29CyR (Yao
and Yao, 1991) for biolistic transformation followed by selection for
cycloheximide resistance. In both VPS8D hairpin constructs,
hairpin expression is under the control of the cadmium-inducibleMTT1 promoter (Shang et al., 2002).