However, rather than express tagged ORFs from their native environments, as in the TAP studies, Ho et al. express tagged ORFs from plasmids containing GAL1 promoters.
Yeast strains used in this study are derived form BY4741 (MAT a his3-Δ1 leu2-Δ0 met15-Δ0 ura3-Δ0): chromosomally integrated strains that express tag-TAP proteins under its own promoter RSC3- TAP, RSC9- TAP, DBF1-TAP and STH1-TAP (from the yeast TAP collection; Open Biosystems) and deletion strains YGM61 (MAT a hog1∷kanMx4), rsc1∷kanMx4, npl6∷kanMx4 and snf6∷kanMx4.
A pGEX4t3-tag plasmid was included to express tagged GST alone, which is necessary for serologic background determinations.
To express tagged Mcl1-S, the TAA stop codon of the above primer was replaced with TTA.
In the following weeks, TRAMP mice over-express Tag and invariably develop spontaneous mouse prostate intraepithelial neoplasia (mPIN), which progress to adenocarcinoma and seminal vesicles, lymph node (LN) and visceral metastases, resembling human PC [26].
B6/K-0 is a kidney cell line, expressing Tag [35].
We showed our protocol to work in two different cell lines of different origin: COS-7, a mammalian cell line expressing TAg endogenously, and CEF (avian) in which we expressed TAg by plasmid transfection.
Over the last 20 years, expressed tag sequencing (EST) [9] has been widely utilized for gene discovery and genome characterization.
For gene expression analysis, the number of expressed tags was counted and then normalized to TPM.
For gene expression analysis, the number of expressed tags was calculated and then normalized to TPM (number of transcripts per million tags); and the differentially expressed tags were used for mapping and annotation.
