Figure 2. The Patch-Methylated L1-LTRGFP-1L Cassette is Expressed at a Low Level in a Subset of Clones.

(A) Orientation-matched, unmethylated (−) and patch-methylated (P) clones were analyzed for GFP expression by flow cytometry along with the RL5 parent cell line (dashed line), which is under-laid with each clone for reference. Median fluorescence values are displayed in the lower right hand corner of each histogram.

(B) The median GFP fluorescence values of RL5, clone 6− and 10 patch-methylated clones analyzed at day 38 post-RMCE are shown. Clone 9P (marked with an asterisk) was chosen for further study.

(C) Quantitative duplex RT-PCR analysis of total RNA isolated from a representative low-expressing patch-methylated clone (9P), the unmethylated control clone 6−, and the RL5 parent line was conducted using primers (horizontal arrows) specific for the spliced transgenic mRNA and the endogenous β-actin gene. Relative expression level was determined by normalizing the transgene-specific amplification product to the β-actin gene product.

(D) Two sets of electronic gates were applied to sort relatively low- and high-expressing cells from clone 9P. A total of 10⁴ viable cells of each subpopulation were sorted and cultured for five days prior to reanalysis by flow cytometry. The median fluorescence values for each sorted pool are shown in the lower right hand corner of each histogram, along with that of the RL5 parent line, and clone 6− for reference.