2.4 Transformation and selection
We transformed the vectors into C. neoformans competent cells
following the operating manual of the Yeast Transformation Kit. However,
we prolonged the resuscitation time of cells transformed with orbicular
vectors to 2-3 h. Every group was named by the plasmid that the cells
transformed, namely group pS-LAC1-A, group pS-LAC1-B, group pS, and
group PKN-LAC1.
After transformation, the selection for group pS-LAC1-A, pS-LAC1-B, and
pS was addressed by YPDA broth with G418 (100 μg/mL) for 10 days,
replacing fresh culture medium every 2-3 days. Then the yeast cells were
maintained in YPDA broth with G418 (50 μg/mL) until harvested on day
14th. After 14 days of selection, the cultures were
centrifuged at 3000 rpm/min for 10 min, washed three times with PBS and
re-suspended in 200 μl sterile ddH20, which shook in 5ml
laccase-inducing media for 1 h and subsequently prepared for the RNA
extraction. In addition, we pipetted 30 μl cell suspension out of 200 μl
re-suspension to plate on the DOPA agar at 30 ℃ for 7 days, aiming to
observe the phenotype of every group. Then we plated the white colonies
to new DOPA agars every 5 days to explore the stability of the RNAi
effect.
After transformation and resuscitation, we washed the cells of group
PNK-LAC1 three times with PBS and re-suspended them in 200 μl sterile
ddH20. Then the re-suspension was evenly coated on the
DOPA agar with hygromycin B (100 μg/mL) and incubated at 30 ℃ for 7
days. Then we repeatedly streak the white colonies on DOPA agar every 5
days to select the stable albino mutant strains. To verify the
transcription of LAC1 , we randomly chose 3 colonies of the albino
strains for RT- qPCR. Firstly, we amplified the colonies in 3 ml YPDA
broths overnight and then in 10 ml YPDA till the log phase growth of
yeast cells (OD600=0.4-0.6). The cells were centrifuged
at 3000 rpm/min for 10 min, washed three times with PBS and re-suspended
in 10 ml laccase-inducing medium for one-hour shaking. Laccase-inducing
cells were finally centrifuged, preparing for the RNA extraction.