Fig. 2. Frequent de novo TE insertions in MEF-derived clonally expanded miPSC lines.
a Experimental design to generate single-cell miPSC clonal lines. Bulk MEFs from a Col1a1-tetO-OKSM mouse (animal I222e2) were purified and reprogrammed by addition of doxycycline. Individual Oct4-GFP positive miPSCs were then isolated via FACS (p0), expanded in serum for 3 passages (p3), and then cultured in serum- or 2i-containing miPSC media for 3 additional passages (p6). DNA was then extracted and analyzed by WGS and mRC-seq for 9 single-cell clones (for both serum and 2i conditions), with 9 further clones analyzed only with mRC-seq. b A full-length de novo B2 inserted and orientated in antisense to intron 15 of Brca1. PolyA tract length is indicated immediately 3′ of the B2. TSDs are depicted as gray arrows flanking the B2. PCR validation (gel pictures shown) involved an empty/filled PCR design where both primers (red arrows) are outside of the B2, generating filled B2 (red arrow) and empty wild-type (blue arrow) products. The B2 was amplified only in either the serum or 2i conditions for the single-cell clone (number 7) where the B2 was detected by genomic analysis, and not in the matched parental MEFs, the C57BL/6 strain, or a single-cell clone (number 16) selected at random. Molecular weight (mw) markers are provided at the left of gel images. c A full-length (3 monomers) L1 A subfamily element inserted de novo antisense to intron 7 of Gpc5. Sequence characteristics and PCR validation results are shown as in panel (b). Promoter monomers are shown as triangles within the L1 5′UTR. d As in (b), except showing an unusual intergenic B1 insertion flanked by both 5′ and 3′ polyA tracts. e A full-length L1 GF inserted de novo antisense to intron 60 of Dmd. PCR validation involved a 5′ genomic primer and a 3′ junction primer (red arrows). As indicated by a gray box with black stripes, the number of monomers is unknown but was >3. f A heavily 5′ truncated, intergenic de novo L1 TF insertion validated by empty/filled PCR, as per (b). Sequence features are annotated as per (b), with the addition of a 34nt 3′ transduction matching a donor L1 TF located on Chromosome 9. PCR using primers (purple arrows) designed to amplify the entire donor L1 indicated it was polymorphic in our colony. Capillary sequencing indicated the donor L1 retained a promoter of 10 monomers and had intact ORFs. g Locus-specific bisulfite sequencing analysis of the donor L1 promoter identified in panel (f), in MEFs, single-cell miPSC clones, and miPSC lines derived from primary cells. top: Assay design and primer locations. CpGs located in the first 3 monomers of the donor L1 were assessed. Orange and gray strokes indicate CpGs covered and not covered, respectively, by sequencing the amplicon with 2×300mer Illumina reads. middle: Mean percentages of donor L1 CpG methylation for 50 non-identical sequences selected at random from each sample. A two-tailed t test was used to compare serum and 2i culture conditions for single-cell miPSC clones 1–4. bottom: Percentages of fully unmethylated (mCpG = 0, filled bars) and heavily unmethylated (0 < mCpG < 5, white bars) reads using the same sequencing data as displayed in the above histogram. h Percentages of fully unmethylated (mCpG = 0) reads corresponding to the donor L1 promoter identified in panel (f), for miPSCs cultured in serum or 2i conditions. Data represent mean methylation ± SD observed for single-cell miPSC clones 1–4 (n = 4 biological replicates of each condition). Significance testing was via two-tailed t test. i, As for (h), except using an assay targeting the L1 TF subfamily monomer. Source data are provided as a Source Data file.