Active retrotransposition by a synthetic L1 element in mice - PubMed (original) (raw)
Active retrotransposition by a synthetic L1 element in mice
Wenfeng An et al. Proc Natl Acad Sci U S A. 2006.
Abstract
Long interspersed element type 1 (L1) retrotransposons are ubiquitous mammalian mobile elements and potential tools for in vivo mutagenesis; however, native L1 elements are relatively inactive in mice when introduced as transgenes. We have previously described a synthetic L1 element, ORFeus, containing two synonymously recoded ORFs relative to mouse L1. It is significantly more active for retrotransposition in cell culture than all native L1 elements tested. To study its activity in vivo, we developed a transgenic mouse model in which ORFeus expression was controlled by a constitutive heterologous promoter, and we established definitive evidence for ORFeus retrotransposition activity both in germ line and somatic tissues. Germ line retrotransposition frequencies resulting in 0.33 insertions per animal are seen among progeny of ORFeus donor element heterozygotes derived from a single founder, representing a >20-fold increase over native L1 elements. We observe somatic transposition events in 100% of the ORFeus donor-containing animals, and an average of 17 different insertions are easily recovered from each animal; modeling suggests that the number of somatic insertions per animal exceeds this number by perhaps several orders of magnitude. Nearly 200 insertions were precisely mapped, and their distribution in the mouse genome appears random relative to transcription units and guanine-cytosine content. The results suggest that ORFeus may be developed into useful tools for in vivo mutagenesis.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Construction and screening of founders with synthetic L1 (ORFeus) transgene. (A) The transgene construct consists of the following sequence elements from 5′ to 3′: (i) a composite CMV IE enhancer/modified chicken β-actin promoter, designated CAG (33); (ii) synthetic L1 ORF1, ORF2, and 5′ portion of 3′ UTR (32); (iii) herpes simplex virus thymidine kinase poly(A) signal (boxed inverted letter A) in antisense orientation to polyadenylate gfp mRNA; (iv) gfp (green block arrow), a modified version of the EGFP coding sequence [the gfp ORF is in antisense orientation relative to L1, and it is interrupted by intron 2 of the human γ-globin gene, which is in sense orientation relative to L1; gfp serves as a retrotransposition indicator gene (18, 34)]; (v) Rous sarcoma virus LTR promoter in antisense orientation relative to L1, which drives gfp transcription (boxed inverted P for promoter); (vi) β-globin poly(A) signal (boxed upright letter A). [numbered arrows above the diagram represent locations of genotyping PCR primers; the region used to generate Southern blotting probes is indicated (purple line)]. (B) Structure of a representative insertion. A typical insertion (i.e., a retrotransposition event derived from the donor transgene) is 5′ truncated, intronless, ends in a poly(A) tail (AAA), and is flanked by target-site duplications (gray triangles) and gDNA sequences (wavy solid lines). (C_–_E) PCR genotyping of founders (F0) and progeny (F1 and N2). PCRs were performed on mouse gDNA using primer pairs complementary to various transgene segments. Primers 1 and 1′ (intron) were used to amplify the indicator gene (C); primers 2 and 1′ (3′ junction) only amplify donor transgene (D); primers 3 and 3′ (3′ end) amplify the 3′ end of transgene sequence common to both donor and insertions (E); hprt primers were used as endogenous PCR controls (F). Mouse DNA samples are identified by corresponding mouse identifications: prefix F for founders and B for progeny from line F210. Kinship among mice is indicated at the top of the gel image. WT, wild-type C57BL/6J; TE, TE buffer; +intron, plasmid that carries indicator cassette with intron; −intron, plasmid with intronless indicator cassette; NTC, PCR mix only; M, 100-bp DNA ladder (New England Biolabs, Ipswich, MA).
Fig. 2.
Estimating new insertion frequency by Southern blot analysis. (A) Schematic of transgene concatemer illustrating expected bands for PstI (P)-digested gDNA. Three copies of transgenes are shown for illustration; the actual copy number was estimated to be 8 to 10 by real-time PCR (data not shown). The probe position is indicated by a purple box. (B) Representative blots are shown for N2 progeny mice derived by backcrossing two F1 mice, B044 and B136, to wild-type C57BL/6J mice (WT), respectively. Numbers below each lane indicate the putative number of new insertions per individual mouse. >, bands comigrating with preexisting bands in F1 donor parents; +, new bands in N2 progeny absent from F1 donor parents; ∗, junctions fragments between donor concatemer and flanking genomic sequence. Group ii mice are highlighted in red. DNA migration positions are indicated (1-kb ladder; New England Biolabs).
Fig. 3.
Insertion profiling by iPCR and insertion-specific PCR. (A) Insertion profiling by iPCR. Insertions were recovered by an iPCR technique (Fig. 7_A_). Six independent PCRs with the identical ligation mix for each mouse are shown. Samples were designated by respective mouse identification and PCR genotyping group. (B) Tissue distribution and inheritance pattern of germ line insertion B131-17. A panel of 15 different tissues from F1 mouse B131 (left) and tail biopsies from 20 of its N2 progeny were amplified by a primer specific to the flanking genomic sequence and an ORFeus primer. (C) Tissue mosaicism and lack of inheritance of a somatic insertion B131-20. M1, 100-bp DNA ladder; M2, 1-kb DNA ladder (New England Biolabs).
Fig. 4.
Chromosomal distribution of mapped insertions. (A) A total of 171 mappable insertions were charted to mouse genome build 36 (short black lines to the right of individual chromosomes). The approximate position of donor concatemer on chromosome 7 is marked (red asterisks). The Y chromosome insertion was mapped to multiple Y chromosome-specific BAC clones. (B) Mapping donor transgene location by FISH. Metaphase spreads of splenocytes from donor-containing mice were probed with fluorescently labeled full-length transgene cDNA probe (green) and subsequently with a whole-chromosome paint probe for chromosome 7 (red). Chromosomes were counterstained with DAPI (blue). (C) Distribution of insertions within annotated genes. The point of integration for every intragenic insertion (a total of 65) was transformed into a percentile value relative to the gene length (from 5′ to 3′) and binned into five-percentile intervals (x axis). Integration frequency was derived by dividing the number of intragenic insertions in each interval by the total number of intragenic insertions (y axis). (D) Distribution of ORFeus insertions relative to local GC content. GC content was determined for a 50-kb region centered at each insertion, and the frequency of integration was plotted as a function of the local GC content (right y axis); as a comparison, the mouse genome was binned as 50-kb segments by guanine-cytosine (GC) content (left y axis).
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