Multiplex transgenesis in cattle via the sleeping beauty transposon system

GARRELS, WIEBKE; TALLURI, THIRUMALA R.; APFELBAUM R.; BOSCH, P.; GRUESO, ESTHER; IVICS, ZOLTAN; KUES, WILFRIED A.

Praga

Congreso; 13th Transgenic Technology Meeting; 2016

International Society for Transgenic Technologies

Cattle are an important model for zoonotic diseases, such abovine spongiform encephalitis and tuberculosis. Geneticengineering can expand the utility of cattle for modellinghuman diseases, for developing novel therapies and for theproduction of recombinant proteins in the udder. However, theefficiencies for the generation of genetically engineered cattleby the available techniques, like pronuclear injection orcloning methods are relative inefficient.Here, we present a highly efficient method for thegeneration of multi transgenic cattle, produced via the SleepingBeauty (SB) transposon system in combination with cytoplasmicplasmid injection (CPI). We generated heteromerictransposon transgenic cattle with ubiquitous expression of theVenus fluorophore, lens-specific expression of the tdTomatoreporter and udder-specific expression of a viral envelopesubunit. The transposase catalyzed gene delivery increased theefficiency of chromosomal integration and favoured singlecopy insertion events into euchromatic regions. Zygotes wereproduced in vitro and were injected via cytoplasmic plasmidinjection. Injected embryos were cultured for 7 days toblastocyst stage in vitro and then screened for Venus fluorescence.Venus positive blastocysts were transferred tosynchronized fosters (n = 6) and after nine moth two transgeniccalves were born. The Venus-transposon containsheterospecific loxP sites, which allows a targeted exchangeof the Venus transgene cassette in a pretested locus against atransgene of choice by transient expression of Cre recombinaseand subsequently the cells can be used for somatic cell nucleartransfer.