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Controlled Insertional Mutagenesis Using a LINE-1 (Orfeus) Gene-Trap

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Controlled Insertional Mutagenesis Using a LINE-1 (Orfeus) Gene-Trap Controlled insertional mutagenesis using a LINE-1 PNAS PLUS (ORFeus) gene-trap mouse model Kathryn A. O’Donnella,b,1,2, Wenfeng Ana,b,3, Christina T. Schruma,b, Sarah J. Wheelanc, and Jef D. Boekea,b,c,1 Departments of aMolecular Biology and Genetics and cOncology, and bHigh Throughput Biology Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Edited* by Fred H. Gage, The Salk Institute for Biological Studies, San Diego, CA, and approved May 30, 2013 (received for review February 7, 2013) A codon-optimized mouse LINE-1 element, ORFeus, exhibits dra- treated with doxycycline. We observed high levels of retro- matically higher retrotransposition frequencies compared with transposition in tissues from double-transgenic mice but not its native long interspersed element 1 counterpart. To establish in control littermates, and the amount of retrotransposition a retrotransposon-mediated mouse model with regulatable and increases with increased doxycycline dose. Induction of the tet- potent mutagenic capabilities, we generated a tetracycline (tet)- ORFeus element with high doses of doxycycline during embryo- regulated ORFeus element harboring a gene-trap cassette. Here, genesis led to a reduced number of double-transgenic mice, we show that mice expressing tet-ORFeus broadly exhibit robust likely due to a significant burden of mutations and embryonic retrotransposition in somatic tissues when treated with doxycy- lethality in these animals. Unexpectedly, a significant percentage cline. Consistent with a significant mutagenic burden, we observed of double-transgenic agouti mice developed white spots, suggest- a reduced number of double transgenic animals when treated with ing that somatic mutations occurred at different times in de- high-level doxycycline during embryogenesis. Transgene induction velopment. Consistent with this, this phenotype is not heritable, in skin resulted in a white spotting phenotype due to somatic and we therefore infer that it occurred somatically in melano- ORFeus-mediated mutations that likely disrupt melanocyte develop- cytes or their precursors. We show that the white spots lack ment. The data suggest a high level of transposition in melanocyte melanocytes, suggesting that the ORFeus element has somat- precursors and consequent mutation of genes important for mela- ically altered a gene(s) involved in melanocyte development, noblast proliferation, differentiation, or migration. These findings proliferation, or migration. With the development and charac- GENETICS reveal the utility of a retrotransposon-based mutagenesis system terization of the tet-ORFeus model complete, we are poised to as an alternative to existing DNA transposon systems. Moreover, use this retrotransposon-based system as a tool for cancer gene breeding these mice to different tet-transactivator/reversible tet- discovery and other forward genetic screens. Moreover, this transactivator lines supports broad functionality of tet-ORFeus system may be useful as a general tool for mutagenesis in mice. because of the potential for dose-dependent, tissue-specific, and temporal-specific mutagenesis. Results Generation of a Conditional L1 Retrotransposon Gene-Trap Element. L1 retrotransposon | tet-promoter | white-spotted phenotype | We generated a conditional synthetic L1 retrotransposon by mus musculus placing the ORFeus element under the control of the tetracy- cline-responsive promoter (TRE) (10, 11). Confirmation of tight ong interspersed element 1 (LINE-1 or L1) retrotransposons Lare an abundant class of mobile genetic sequences that Significance constitute ∼17% of the human and mouse genomes (1, 2). In contrast to DNA transposons, L1 elements retrotranspose Transposons are powerful tools widely used for insertional using a copy-and-paste mechanism in which they are first tran- mutagenesis screens because of the straightforward identi- scribed into an RNA intermediate before insertion into a new fication of transposon-induced mutations. We developed a genomic location (3–5). Because L1 retrotransposons are not conditionally regulated long interspersed element 1 [tetra- excised from genomic DNA (gDNA), the donor elements are cycline (tet)-ORFeus] retrotransposon mouse model that may stable. Furthermore, studies have revealed that L1 elements ex- be used for random mutagenesis and mechanistic studies. High- hibit relatively unbiased insertion-site selection (6). These findings level expression of the retrotransposon during development suggest that retrotransposons may be efficient for utilization in results in embryonic lethality due to a significant burden of genome-wide insertional mutagenesis screens. insertions. At lower levels, transgene induction results in a so- A synthetic mouse L1 element was recently constructed by matic spotting phenotype due to mutations that likely disrupt altering the nucleic acid sequence without changing the amino melanocyte development, suggesting high activity of tet-ORFeus acid sequence of L1-encoded proteins. These optimized ele- in melanocyte precursors. This model may be used to identify ments abolished transcription-inhibitory sequences and resulted genes and pathways involved in diverse biological processes. in a >200-fold increase in retrotransposition frequencies when tested in cell culture (7). Subsequently, we generated a mouse Author contributions: K.A.O., W.A., and J.D.B. designed research; K.A.O. and C.T.S. per- formed research; K.A.O., S.J.W., and J.D.B. analyzed data; and K.A.O. and J.D.B. wrote model expressing this element, ORFeus, which demonstrated in the paper. vivo retrotransposition activity when driven by a constitutive pro- The authors declare no conflict of interest. moter (8). This approach was further modified through the acti- *This Direct Submission article had a prearranged editor. vation of ORFeus using Cre-Lox recombination technology (9). To generate a chemically regulated L1 mouse model with potent Freely available online through the PNAS open access option. 1 mutagenic capabilities, we generated a tetracycline (tet)-regulated To whom correspondence may be addressed. E-mail: kathryn.odonnell@UTSouthwestern. edu or [email protected]. ORFeus element harboring a gene-trap cassette designed to trun- 2Present address: Department of Molecular Biology, UT Southwestern Medical Center, cate target transcripts or activate downstream transcription, de- Dallas, TX 75390-9148. pending on the orientation of the gene trap. 3Present address: School of Molecular Biosciences and Center for Reproductive Biology, We demonstrate that, when mice harboring a tet-ORFeus Washington State University, Pullman, WA 99164. gene-trap transgene are bred with a reversible tet-transactivator This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (rtTA) line, double-transgenic progeny express ORFeus only when 1073/pnas.1302504110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1302504110 PNAS Early Edition | 1of8 Downloaded by guest on September 27, 2021 tet-regulated control of ORFeus expression was obtained by was designed to disrupt gene function in several ways (Fig. S1A). RNA blot analysis in Tet-ON and Tet-OFF HeLa cells (Fig. 1 A The gene trap contains a splice acceptor followed by a poly- and B). We also compared the expression of ORFeus when adenylation (polyA) signal. When the element transposes into an driven by the TRE promoter versus the constitutive cytomega- intron, the host gene may splice to the gene trap. Resulting use lovirus early enhancer/chicken beta-actin (CAG) promoter and of the gene-trap polyA signal will truncate the mRNA, likely found that mRNA levels were similar (Fig. 1C). We further resulting in a loss-of-function mutation (Fig. S1B). The gene-trap assessed the retrotransposition frequency of the tet-ORFeus el- cassette also contains a long terminal repeat promoter/enhancer ement in a standard cell culture retrotransposition assay. In this element that is designed to activate gene expression when trans- system, a functional L1 element is marked with a retrotrans- posed upstream or within a gene, resulting in the activation of position indicator reporter (5). In this case, we used an indicator either a full-length or a truncated protein, respectively (Fig. S1C). gene conferring resistance to blasticidin. Intron removal during In addition, the gene trap harbors a small chimeric intron, which splicing of the L1 transcript restores function to a blasticidin re- provides a simple assay for splicing and, by extension, for retro- sistance gene encoded on the opposite strand. Quantification of transposition of the element. Removal of this intron is a signal that blasticidin-resistant colonies demonstrated that the tet-ORFeus retrotransposition has occurred because these elements proceed element retrotransposed in a doxycycline-dependent manner through an RNA intermediate; thus, a simple PCR-based assay and at slightly higher frequency than the CAG-ORFeus element for intron removal gives a quick readout of whether a given cell or mouse line is active for retrotransposition. RT-PCR studies (Fig. 1D). fi To generate a conditional ORFeus element that also serves as documented high-level expression and ef cient splicing of the a mutagen, we engineered the tet-ORFeus transgene to contain chimeric intron upon induction of the tet-ORFeus gene-trap a gene-trap cassette in its 3′ untranslated region. The gene trap element in tet-OFF HeLa cells (Fig. S1D). We next generated transgenic mice harboring the tet-ORFeus gene-trap element by pronuclear
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