Fig. 5

New L1 integrants in mouse embryonic stem (mES) cells undergo dense cytosine methylation. Dense cytosine methylation at new L1 integrants in mouse ES cells was revealed by bisulfite sequencing. Initially, Bruce 4 cells were transfected with pJH435, encoding an inactivated L1 ORFeus donor element marked with GFPuv-AI reporter regulated by a respiratory syncytial virus (RSV) promoter. Upon activation of L1 donor expression by transient infection of the culture using adenoviral Cre recombinase, individual colonies were picked and cultured on feeder cells for > 2 months. Of these mES subclones, most harbored newly retrotransposed L1 reporter integrants, as shown by a PCR-based assay documenting spliced integrated copies of the reporter GFPuv-AI (not shown). Their cytosine methylation status was assessed either in bulk or at individual loci using bisulfite sequencing. a For mES subclone 1B6-A07, we used primers DES3301 x DES3314, which anneal within the gene encoding GFPuv. This PCR amplicon does not cross the AI splice site, so unspliced donor L1 sequences also can be amplified. b For mES subclone 1B6-A08, primers DES3298 x DES3299 were used. c For mES clones 1B06/B02, 1C6 and 2D4, primers DES3321 x DES3322 were used to assay 15 CpG dinucleotides in a 234 nt amplicon across the GFPuv-AI splice junction