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.

Going non-viral: theSleeping Beautytransposon system breaks on through to the clinical side

“…In case of a plasmid vector carrying the transposon unit, this means shortening the plasmid backbone. By placing the transposon TIRs next to one another, bacterial artificial chromosomes (BACs) over 100 kb in length have been demonstrated to transpose at reasonable efficiencies in human ES cells (Rostovskaya et al, 2012;Rostovskaya et al, 2013). The second mechanism limiting transposition of large constructs is a suicidal transpositional mechanism called autointegration .…”

Induction of Potent CD8 T Cell Cytotoxicity by Specific Targeting of Antigen to Cross-Presenting Dendritic Cells In Vivo via Murine or Human XCR1

“…BAC DNA was prepared from bacterial culture grown at 30˚C using the Nucleobond BAC 100 kit (Macherey-Nagel), digested with MluI/AscI for removing the bacterial backbone, purified by Sepharose CL-4B chromatography (22), and injected (1-4 ng/ml) into the pronucleus of C57BL/6J fertilized oocytes. Offspring was genotyped using the primer pair 59-GCACGGTCAAGCTCATCTTCGCC-39 and 59-ATGCTCCTTCCAGGC-CCGCT-39.…”

piggyBacTransposon

“…The cargo capacity of the piggyBac transposon is fairly large; transposons with a cargo of up to 10 kb can be mobilized without losing transposition efficiency (62). Recently, it has been shown that piggyBac can transpose bacterial artificial chromosomes (BACs), which are 150 to 300 kb in length, in mouse and human pluripotent stem cells (50,63) and in mouse zygotes (64). In the best result in the zygote injection, 45% of F0 mice carried a BAC transgene and most of them have transposon signature (64).…”