PMC:3763561 / 1007-3344 JSONTXT

{"project":"2_test","denotations":[{"id":"23847100-22124480-77033131","span":{"begin":187,"end":188},"obj":"22124480"},{"id":"23847100-21885773-77033132","span":{"begin":205,"end":206},"obj":"21885773"},{"id":"23847100-22722847-77033133","span":{"begin":236,"end":237},"obj":"22722847"},{"id":"23847100-20107439-77033134","span":{"begin":282,"end":283},"obj":"20107439"},{"id":"23847100-20418862-77033135","span":{"begin":324,"end":325},"obj":"20418862"},{"id":"23847100-21081918-77033136","span":{"begin":438,"end":439},"obj":"21081918"},{"id":"23847100-22422837-77033137","span":{"begin":440,"end":441},"obj":"22422837"},{"id":"23847100-21750718-77033138","span":{"begin":1008,"end":1009},"obj":"21750718"},{"id":"23847100-21081918-77033139","span":{"begin":1163,"end":1164},"obj":"21081918"},{"id":"23847100-17293868-77033140","span":{"begin":1165,"end":1166},"obj":"17293868"},{"id":"23847100-19073939-77033141","span":{"begin":1187,"end":1189},"obj":"19073939"},{"id":"23847100-22422837-77033142","span":{"begin":1227,"end":1228},"obj":"22422837"},{"id":"23847100-17945255-77033143","span":{"begin":1540,"end":1542},"obj":"17945255"}],"text":"The ability to introduce into mammalian cells genetic circuits that contain multiple transcription units (TUs) is of great interest for a variety of applications including biotechnology (1), gene therapy (2), systems/synthetic biology (3) and reprogramming cell fate and functions (4), as well as basic biological research (5). Encoding such multi-TU gene circuits on single vectors offers several advantages over using separate vectors (6,7), for example, to improve correlation in gene expression between the different circuit elements and for an integration of the entire circuit into a single genomic locus. However, the construction of such large single-vector circuits is challenging because of long and/or repetitive sequences and the need for genetic elements that impart robust expression in mammalian cells. Existing DNA assembly methods are often not well suited for manipulating large collections of mammalian sequences. For example, methods that rely on the use of Type IIs restriction enzymes (8) can be problematic because these restriction sites occur frequently in mammalian promoters and genes. Other methods require multiple rounds of cloning (6,9), cloning in yeast (10) or polymerase chain reaction (PCR) (7). With PCR, the precision of even high-fidelity polymerases is insufficient for reliable and error-free large-scale amplification (Supplementary Table S1). Furthermore, multi-TU gene circuits lacking insulating elements suffer from transcriptional interference and are significantly hampered in their function (11). To address these issues, we developed a new framework for quick and reliable assembly of functional complex mammalian gene circuits. Here, we describe in detail the components, steps and mechanisms underlying the framework. We demonstrate efficient and robust construction of circuits with various sizes and number of assembled parts, and show that assembly works well despite repetitive sequences present in some of the parts. The resulting gene circuits were functionally assessed in transfection as well as stable genomic integration and behaved according to their predicted phenotypes. The framework described here can also prove to be valuable for building large-scale mammalian genetic module libraries, and is well suited for generation of stable cell lines with multielement circuits."}