title: Modular assembly of transposon integratable multigene vectors using RecWay assembly.
abstract: Studying complex biological processes such as cancer development, stem cell induction and transdifferentiation requires the modulation of multiple genes or pathways at one time in a single cell. Herein, we describe straightforward methods for rapid and efficient assembly of bacterial marker free multigene cassettes containing up to six complementary DNAs/short hairpin RNAs. We have termed this method RecWay assembly, as it makes use of both Cre recombinase and the commercially available Gateway cloning system. Further, because RecWay assembly uses truly modular components, it allows for the generation of randomly assembled multigene vector libraries. These multigene vectors are integratable, and later excisable, using the highly efficient piggyBac (PB) DNA transposon system. Moreover, we have dramatically improved the expression of stably integrated multigene vectors by incorporation of insulator elements to prevent promoter interference seen with multigene vectors. We demonstrate that insulated multigene PB transposons can stably integrate and faithfully express up to five fluorescent proteins and the puromycin-thymidine kinase resistance gene in vitro, with up to 70-fold higher gene expression compared with analogous uninsulated vectors. RecWay assembly of multigene transposon vectors allows for widely applicable modelling of highly complex biological processes and can be easily performed by other research laboratories.
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA. mori0164@umn.edu
authors: Branden S
authors: BS
authors: Moriarity
authors: Eric P
authors: EP
authors: Rahrmann
authors: Vincent W
authors: VW
authors: Keng
authors: Luke S
authors: LS
authors: Manlove
authors: Dominic A
authors: DA
authors: Beckmann
authors: Natalie K
authors: NK
authors: Wolf
authors: Touba
authors: T
authors: Khurshid
authors: Jason B
authors: JB
authors: Bell
authors: David A
authors: DA
authors: Largaespada
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004251:DNA Transposable Elements; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D019426:Integrases / Q000378:metabolism; D051379:Mice; D009374:Neoplasms, Experimental / Q000235:genetics; D034741:RNA, Small Interfering / Q000378:metabolism; D014162:Transfection
chemical_list: D004251:DNA Transposable Elements; D018076:DNA, Complementary; D034741:RNA, Small Interfering; C045073:Cre recombinase; D019426:Integrases