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.