Identification and re-addressing of a transcriptionally permissive locus in the porcine genome

Abstract

Recently, we established the Sleeping Beauty transposon system for germ line competent transgenesis in the pig. Here, we extend this approach to re-target a transposon-tagged locus for a site-specific gene knock-in, and generated a syngeneic cohort of piglets carrying either the original transposon or the re-targeted event. A Cre-loxP-mediated cassette exchange of the tagging transposon with a different reporter gene was performed, followed by flow cytometric sorting and somatic cell nuclear transfer of recombined cells. In parallel, the original cells were employed in somatic cell nuclear transfer to generate clone siblings, thereby resulting in a clone cohort of piglets carrying different reporter transposons at an identical chromosomal location. Importantly, this strategy supersedes the need for an antibiotic selection marker. This approach expands the arsenal of genome engineering technologies in domestic animals, and will facilitate the development of large animal models for human diseases. Potentially, the syngeneic cohort of pigs will be instrumental for vital tracking of transplanted cells in pre-clinical assessments of novel cell therapies.

Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP

“…Our study and a recent report demonstrated the potentiality of Cre/LoxP in engineering livestock. Garrels et al took advantage of Cre/LoxP-mediated cassette exchange to generate syngeneic cohort of pigs carrying different reporter transposons at an identical chromosomal location with a comparable efficiency as we showed in this study 26 . It is safe to say that Cre/LoxP technology still has very important applications in genetically modified livestock owning to its highly efficient re-targeting capability like site-specific excision and cassette exchange.…”

Expression of Active Fluorophore Proteins in the Milk of Transgenic Pigs Bypassing the Secretory Pathway

“…Here, we showed that ubiquitously expressed Venus and mCherry fluorophore proteins could be harvested at large scale in the milk of transposon transgenic sows. Genomic integration of transgenes by the SB transposase into transcriptionally permissive loci allows the reduction of the number of experimental animals and contributes to robust transgene expression levels 65 . Importantly, the SB system has been established for successful transposition in a range of mammals suitable for milking 35 66 67 .…”

“…Briefly, the Venus positive porcine line has been generated by co-injection of pT2/RMCE-Venus transposon and pCMV-SB100X transposase plasmids into the cytoplasm of porcine zygotes. The mCherry positive porcine line has been developed by a targeted recombination-mediated cassette exchange (RMCE) of Venus against mCherry cDNAs 34 65 .…”

Sleeping Beautytransposition: from biology to applications

“…A powerful strategy could be used to express a series of expression cassettes from the same genomic locus by cassette exchange using Cre or FLP recombinases (Garrels et al, 2016a;Grabundzija et al, 2013).…”

“…This feature also helps to establish tissue-specific transgenic models (Katter et al, 2013). Combining SB transgenesis with recombinase-mediated cassette exchange could be employed to test multiple expression cassettes at a single, well-characterized genomic locus (Garrels et al, 2016a). Transgenic animals stably expressing specific markers could be used to monitor dynamic processes and establish various drug-testing schemes (Szebenyi et al, 2015).…”