title: Transposon mediated BAC transgenesis via pronuclear injection of mouse zygotes.
abstract: Pronuclear microinjection of bacterial artificial chromosomes (BACs) is the preferred way to generate transgenic mice because the transgene accurately recapitulates expression of the endogenous gene. However, the method is demanding and the integrity and copy number of the BAC transgene is difficult to control. Here, we describe a simpler pronuclear injection method that relies on transposition to introduce full-length BACs into the mouse genome. The bacterial backbone of a hPAX6-GFP reporter BAC was retrofitted with PiggyBac transposon inverted terminal repeats and co-injected with PiggyBac transposase mRNA. Both the frequency of transgenic founders as well as intact, full-length, single copy integrations were increased. Transposition was determined by a rapid PCR screen for a transpositional signature and confirmation by splinkerette sequencing to show that the BACs were integrated as a single copy either in one or two different genomic sites. BAC transposons displayed improved functional accuracy over random integrants as evaluated by expression of the hPAX6-GFP reporter in embryonic neural tube and absence of ectopic expression. This method involves less work to achieve increased frequencies of both transgenesis and single copy, full-length integrations. These advantages are not only relevant to rodents but also for transgenesis in all systems.
authors.affiliation: Stem Cell Engineering, Biotechnology Center, Technische Universitaet Dresden, Dresden, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Rostovskaya
authors.forename: Ronald
authors.initials: R
authors.lastname: Naumann
authors.forename: Jun
authors.initials: J
authors.lastname: Fu
authors.forename: Mandy
authors.initials: M
authors.lastname: Obst
authors.forename: Doris
authors.initials: D
authors.lastname: Mueller
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Anastassiadis
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial; D004251:DNA Transposable Elements / Q000235:genetics*; D005136:Eye Proteins / Q000235:genetics; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D008967:Molecular Biology / Q000379:methods; D054259:Neural Tube; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics; D016133:Polymerase Chain Reaction; D012097:Repressor Proteins / Q000235:genetics; D019895:Transposases / Q000235:genetics; D015053:Zygote
chemical_list: D004251:DNA Transposable Elements; D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; D019895:Transposases