Transposon-mediated BAC transgenesis in human ES cells

Maria Rostovskaya 2 Jun Fu 1 Mandy Obst 2 Isabell Baer 1 Stefanie Weidlich 2 Hailong Wang 1 Andrew J. H. Smith 0 Konstantinos Anastassiadis 2 A. Francis Stewart 1 0 Genome Engineering Group, Institute for Stem Cell Research, MRC Centre for Regenerative Medicine, University of Edinburgh , Edinburgh , EH16 4UU, UK 1 Genomics , BioInnovationsZentrum, Technische Universitaet Dresden , Dresden 01307, Germany 2 Stem Cell Engineering Transgenesis is a cornerstone of molecular biology. The ability to integrate a specifically engineered piece of DNA into the genome of a living system is fundamental to our efforts to understand life and exploit its implications for medicine, nanotechnology and bioprospecting. However, transgenesis has been hampered by position effects and multi-copy integration problems, which are mainly due to the use of small, plasmid-based transgenes. Large transgenes based on native genomic regions cloned into bacterial artificial chromosomes (BACs) circumvent these problems but are prone to fragmentation. Herein, we report that contrary to widely held notions, large BAC-sized constructs do not prohibit transposition. We also report the first reliable method for BAC transgenesis in human embryonic stem cells (hESCs). The PiggyBac or Sleeping Beauty transposon inverted repeats were integrated into BAC vectors by recombineering, followed by co-lipofection with the corresponding transposase in hESCs to generate robust fluorescent protein reporter lines for OCT4, NANOG, GATA4 and PAX6. BAC transposition delivers several advantages, including increased frequencies of single-copy, full-length integration, which will be useful in all transgenic systems but especially in difficult venues like hESCs. - Early work on transgenesis in animals and cell lines invariably used small transgenes, which only rarely achieved the intended expression pattern due mainly to position effects exerted by the genomic integration site or concatamerization. These major problems have been circumvented by the use of large transgenes such as bacterial artificial chromosomes (BACs), which carry intact genomic regions and often deliver the expected expression pattern precisely (1). Due to their large size, BACs can accommodate complete genes including all cis-regulatory elements in their native configuration. Consequently, most BAC transgenes are indifferent to position effects and often deliver expression levels in proportion to the transgene copy number. Many BAC libraries have been annotated onto genome browsers and are readily available from genome resource providers such as CHORI (www.chori.org). Furthermore, BACs can be readily modified and mutated using recombineering (25). These advantages have promoted BACs to the forefront as transgenic tools and now BAC transgenesis has been successfully applied to produce a variety of transgenic animals, such as mice, rats, zebrafish and flies (69), as well as for studies of gene function, molecular complementation of mutations, identification of distant regulatory elements and analysis of gene dosage, among other applications (1,1013). Because they often recapitulate expression patterns precisely, BAC transgenes are also widely used to create gene expression reporters for studies during development and differentiation. Human embryonic stem cells (hESCs) (14) provide an essential venue for studies of human development and disease that complements work with model systems such as the mouse. Like mouse ESCs (mESCs), they can be differentiated in culture to recapitulate aspects of human embryology and to serve as paradigms for future medicine with cellular therapies. However, they are difficult to manipulate genetically, particularly for gene targeting (1517). The work reported here began with our efforts to create stable hESC reporter lines based on fluorescent protein expression driven by stage- and lineage-specific promoters. Although we were able to create an OCT4-GFP reporter line by gene targeting (data not shown), the efficiency of homologous recombination in hESCs is low (15,16) and our attempts to generate a knock-in for lineage-specific genes have not been successful. On the other hand, randomly integrated retroviral and small transgenes often undergo transcriptional silencing in hESCs (1719). Consequently, we were attracted by the advantages of BAC transgenesis and used the only published method for BAC transgenesis in hESCs, which is based on nucleofection (20). Unexpectedly, transgene silencing was consistently observed, which we correlated with consistent failures to obtain integrations of full-length BAC transgenes. To solve the problem of BAC fragmentation, we explored the possibility that transposition could be used to integrate full-length BAC transgenes. DNA transposons are mobile elements that contain inverted terminal repeats (ITRs), which are recognition sites for a transposase that cuts at the outside end of the inverted repeats and moves the excised DNA into a new site. Transposons have been used for insertional mutagenesis and gene transfer in many model organisms. However, applications in vertebrates were impeded due to the lack of active transposons until Tol2 was isolated from the Japanese Medaka fish Oryzias latipes (21,22) and Sleeping Beauty (SB) was reactivated from the salmon genome by the elimination of phylogenetically identified mutations (23,24). In 2005, PiggyBac transposon isolated from the cabbage looper moth Trichoplusia ni was reported to be active in mammalian cells including mouse and human (25). Consequently, several options for transposition in fish, mouse and human cells are now available. In particular, SB and PiggyBac appear most useful (2631) and increased activity variants of both have been recently identified (32). Notably, transposase-mediated transgenesis has been used in cells that are difficult to transfect including human haematopoietic stem cells (32,33) and hESCs (3436). Consequently, we were encouraged to examine whether BAC transgenesis in hESCs could be facilitated by transposition. However, transposons appear to have severe size limitations (37), which have limited their use for large transgenes. During attempts to integrate large (up to 60 kb) transgenes into Myxococcus and Pseudomonas prokaryotic hosts, we encountered problems with fragmentation, which we solved by use of transposition (38). Furthermore, Tol2 transposition has been used to integrate a 66 kb transgene into zebrafish and mouse genomes (39). These studies indicate that fears about the size limitations of transposons may be misguided. Herein, we show that transposition can be applied to integrate full-length BACs larger than 150 kb into hESCs, which has implications for BAC transgenesis in general and particularly in systems that are difficult to work with. MATERIALS AND METHODS Generation of large reporter constructs and BAC reporters The large constructs were made by subcloning from the respective BACs a region (...truncated)