Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double‐transgenic pigs

Abstract

The applicability of tightly regulated transgenesis in domesticated animals is severely hampered by the present lack of knowledge of regulatory mechanisms and the long generation intervals. To capitalize on the tightly controlled expression of mammalian genes made possible by using prokaryotic control elements, we have used a single-step transduction to introduce an autoregulative tetracycline-responsive bicistronic expression cassette (NTA) into transgenic pigs. Transgenic pigs carrying one NTA cassette showed a mosaic transgene expression restricted to single muscle fibers. In contrast, crossbred animals carrying two NTA cassettes with different transgenes, revealed a broad tissue-independent and tightly regulated expression of one cassette, but not of the other one. The expression pattern correlated inversely with the methylation status of the NTA transcription start sites indicating epigenetic silencing of one NTA cassette. This first approach on tetracycline regulated transgene expression in farm animals will be valuable for developing precisely controlled expression systems for transgenes in large animals relevant for biomedical and agricultural biotechnology.

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

“…Humanized pig models include those for retinitis pigmentosa 6 , cystic fibrosis 7 , Alzheimer´s disease 8 , Huntington´s disease 9 , familial adenomatous polyposis 10 , and immunodeficiency 11 . However, transgenesis in the pig, most commonly achieved by pronuclear DNA injection (PNI) or by somatic cell nuclear transfer (SCNT), is an inefficient and expensive process, hampered by poor predictability of levels and patterns of transgene expression 2,3,[12][13][14] . Gene targeting by homologous recombination is extremely inefficient in porcine somatic cells 3 , and porcine embryonic stem cells (ES) have not yet been established 15 .…”

Derivation and Characterization ofSleeping BeautyTransposon-Mediated Porcine Induced Pluripotent Stem Cells

“…An apparent advantage of SB-catalyzed transgenesis is the preferential integration into transcriptionally permissive genomic loci [36]. In contrast, random transgenesis frequently resulted in silenced transgenes in the pig [37]. Importantly, the SB transposase has a close-to-random insertion profile in mammalian genomes [33].…”

Unexpected Expression Pattern of Tetracycline-Regulated Transgenes in Mice

“…Failure of tet-based strategies in vivo has also been reported. This has been related either to defective tTA expression (Bö ger and Gruss 1999; Fedorov et al 2001;Lee et al 2006) or to epigenetic repression on the tet-promoter activity ( Janicki et al 2004;Pankiewicz et al 2005;Kues et al 2006), in addition to undesired interactions of eukaryotic cell factors with the tetpromoter sequences (Rang and Will 2000;Gould and Chernajovsky 2004).…”