Efficient Genome-Wide First-Generation Phenotypic Screening System in Mice Using the Piggybac Transposon
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Efficient genome-wide first-generation phenotypic screening system in mice using the piggyBac transposon Hao Changa,b,c,1, Yukun Pana,b,1,2, Sean Landrettea,b, Sheng Dinga,b, Dong Yanga,c, Lufang Liua,b, Lei Tiana,b, Hongyan Chaid, Peining Lid, Da-Ming Lia,b,c, and Tian Xua,b,c,3 aDepartment of Genetics, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536; bHoward Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536; cSchool of Life Sciences, Westlake Institute for Advanced Study, Westlake University, 310024 Hangzhou, China; and dLaboratory of Clinical Cytogenetics and Genomics, Department of Genetics, Yale School of Medicine, New Haven, CT 06536 Edited by Norbert Perrimon, Howard Hughes Medical Institute, Boston, MA, and approved August 1, 2019 (received for review April 18, 2019) Genome-wide phenotypic screens provide an unbiased way to tetranucleotide target sites, has been modified to generate a bi- identify genes involved in particular biological traits, and have nary transposon system for mammalian cells and mammals. The been widely used in lower model organisms. However, cost and system contains a nonautonomous PB transposon cassette to de- time have limited the utility of such screens to address biological liver the exogenous genes of interest flanked by the PB inverted and disease questions in mammals. Here we report a highly efficient repeat sequences, and a transgene expressing the PB transposase piggyBac (PB) transposon-based first-generation (F1) dominant enzyme (PBase) for inducing transposition in the germline. screening system in mice that enables an individual investigator to Transposition occurs when PBase binds to the inverted repeat se- conduct a genome-wide phenotypic screen within a year with fewer than 300 cages. The PB screening system uses visually trackable quences of the PB transposon, initiating excision and reintegration transposons to induce both gain- and loss-of-function mutations into another locus. The binary PB system can efficiently transpose and generates genome-wide distributed new insertions in more in mice and human cells (9). Somatic mutagenesis using multiple than 55% of F1 progeny. Using this system, we successfully con- copies of Sleeping beauty (SB)orPB transposons to generate GENETICS ducted a pilot F1 screen and identified 5 growth retardation muta- dominant mutations have facilitated cancer gene discovery (10– + tions. One of these mutants, a Six1/4PB/ mutant, revealed a role in 13), highlighting the potential to modify this strategy for germline milk intake behavior. The mutant animals exhibit abnormalities in mutagenesis. Indeed, an SB germline system has been developed nipple recognition and milk ingestion, as well as developmental for region-specific saturation screens (14). A comparable system defects in cranial nerves V, IX, and X. This PB F1 screening system for genome-wide mutagenesis is needed. The PB transposon has offers individual laboratories unprecedented opportunities to con- been shown to have higher jumping efficiency and less-severe local duct affordable genome-wide phenotypic screens for deciphering hopping than SB in cultured mouse ES cells (15), thus holding the genetic basis of mammalian biology and disease pathogenesis. promise for affordable genome-wide in vivo germline mutagenesis screens in mice. piggyBac transposon | genome-wide screen | growth retardation | six1 | six4 Significance enome-wide phenotypic screens in mammals are critical for identifying the genes and pathways that are essential for Genome-wide, phenotype-driven mutagenesis in animal models G ’ human health and altered in diseases, but costs associated with could provide an unbiased way to decode a gene s functions and the generation of mutant animals and mapping of mutations its role in diseases. Here we have generated a piggyBac (PB) limit the practicality of current approaches. Traditionally, mouse transposon-based first-generation (F1) dominant screening sys- mutants have been generated from embryonic stem (ES) cells tem in mice, which provides unprecedented opportunities to with specific genes knocked out by homologous recombination conduct a highly efficient and affordable genome-wide pheno- (1, 2). CRISPR/Cas9 is a faster and more efficient technology to typic screen for an individual investigator in a single laboratory. generate mutations in individual genes (3). However, both ap- Using this system, we carried out an F1 dominant screen for growth retardation and discovered 5 isolated mutants that carry proaches remain prohibitively expensive for generating and transposon insertions hitting the genes Rin2, Rbm39, Mll, Zeb2, maintaining genome-wide mouse mutant libraries for phenotypic + and Six1/4.TheSix1/4PB/ mutant animals exhibit abnormalities screening. In contrast to gene-by-gene approaches, chemical mu- in nipple recognition and milk ingestion during the breastfeed- tagens, such as ENU, randomly generate point mutations and are ing period and also exhibit cranial nerve defects. highly potent in mice (4). While recessive screens require a costly 3 generations of breeding, first-generation (F1) dominant screens Author contributions: H. Chang, Y.P., D.-M.L., and T.X. designed research; H. Chang, Y.P., directly assay the progeny of mutagenized animals, reducing cost S.D., L.L., L.T., and D.-M.L. performed research; H. Chang, Y.P., S.L., H. Chai, P.L., and D.-M.L. and time. Genome-scale ENU F1 screens have been conducted contributed new reagents/analytic tools; H. Chang, Y.P., D.Y., and T.X. analyzed data; and for gross morphological, neurological, and immunological defects. H. Chang, Y.P., and T.X. wrote the paper. Key regulatory genes involved in colorectal cancer and circadian The authors declare no conflict of interest. rhythm have also been identified in dominant screens in mice (5– This article is a PNAS Direct Submission. 8). However, ENU F1 screens are still limited by the costs asso- Published under the PNAS license. ciated with the back-crosses and sequencing required for map- 1H. Chang and Y.P. contributed equally to this work. ping mutations. Alternative approaches that make genome-wide 2Present address: Yeda Research Center for Model Organisms, Taizhou, 318000 Zhejiang, screens affordable to individual investigators are highly desirable. China. Transposon insertional mutagenesis is an attractive alternative 3To whom correspondence may be addressed. Email: [email protected]. approach for genome-wide phenotypic screens in mammals. The This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. DNA “cut and paste” piggyBac (PB) transposon from the cabbage 1073/pnas.1906354116/-/DCSupplemental. looper moth Trichoplusia ni, which favorably inserts at TTAA Published online August 26, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1906354116 PNAS | September 10, 2019 | vol. 116 | no. 37 | 18507–18516 Downloaded by guest on September 26, 2021 Here, we developed a highly efficient F1 dominant screening we developed a mutagenic PB construct that includes 4 important system in mice. The new PB system utilizes multiple copies of features: 1) A strong promoter for gene overexpression that can be visually trackable transposons to generate genome-wide muta- conditionally regulated; 2) a terminator for disrupting transcrip- tions in 55.2% of F1 progeny. Therefore, we estimate that this tion; 3) a Katushka red fluorescent protein (Kat) transgene (16); system could allow individual laboratories to perform a genome- and 4) a codon-optimized luciferase gene (luc2) under the control wide phenotypic F1 screen in mice within a year and with 300 cages of a Ubiquitin C (UBC) promoter (Fig. 1A) (12), which is dis- in total. Using this system, we carried out an F1 dominant screen rupted by the insertion of the PB construct. When PBase is for growth retardation and identified both gain- and loss-of- expressed to mobilize PB for mutagenesis, the luc2 gene activity is function alleles. Both intrauterine growth retardation (IUGR) restored as the PB insertion is excised (SI Appendix, Fig. S2A). and postnatal growth retardation (PGR) phenotypes were ob- This construct, named PB[mut-Kat], can induce both gain- and served in the 5 isolated mutants that carry transposon insertions loss-of-function mutations, visually track mutants, and report hitting the endocytosis regulator Rin2, the steroid hormone re- mutagenesis activity. ceptor coactivator Rbm39, the histone methyltransferase Mll,and High mutagenesis efficiency is required for a cost-effective the homeobox transcriptional factors Zeb2 and Six1/4. Altogether, screening system. Mobilizing a single PB transposon in the mouse our work demonstrates the applicability of our system for cost and germline is not sufficient to support a genome-wide screen (9). time-effective phenotypic screens. Increasing the copy number of the mutagenic transposon could improve efficiency, while too many copies of the transposon could Results cause lethality and tumorigenesis (11, 12). We therefore generated Development of a Highly Efficient F1 Mutagenesis Screening System six different mouse lines with copy number of the PB[mut-Kat] in Mice. The F1 mutagenesis system was designed as a binary PB transposon ranging from 1 to 22 (SI Appendix, Table S1). In order system (Fig. 1): A founder line carrying nonautonomous PB trans- to identify a transgenic line able to induce mutations in the poson constructs and a Jump-Starter line carrying a PBase transgene germline without