Assessment of Fecundity and Germ Line Transmission in Two Transgenic Pig Lines Produced by Sleeping Beauty Transposition

Abstract

Recently, we described a simplified injection method for producing transgenic pigs using a non-autonomous Sleeping Beauty transposon system. The founder animals showed ubiquitous expression of the Venus fluorophore in almost all cell types. To assess, whether expression of the reporter fluorophore affects animal welfare or fecundity, we analyzed reproductive parameters of two founder boars, germ line transmission, and organ and cell specific transgene expression in animals of the F1 and F2 generation. Molecular analysis of ejaculated sperm cells suggested three monomeric integrations of the Venus transposon in both founders. To test germ line transmission of the three monomeric transposon integrations, wild-type sows were artificially inseminated. The offspring were nursed to sexual maturity and hemizygous lines were established. A clear segregation of the monomeric transposons following the Mendelian rules was observed in the F1 and F2 offspring. Apparently, almost all somatic cells, as well as oocytes and spermatozoa, expressed the Venus fluorophore at cell-type specific levels. No detrimental effects of Venus expression on animal health or fecundity were found. Importantly, all hemizygous lines expressed the fluorophore in comparable levels, and no case of transgene silencing or variegated expression was found after germ line transmission, suggesting that the insertions occurred at transcriptionally permissive loci. The results show that Sleeping Beauty transposase-catalyzed transposition is a promising approach for stable genetic modification of the pig genome.

Derivation and Characterization of Bovine Induced Pluripotent Stem Cells by Transposon-Mediated Reprogramming

“…Venus is a yellow-shifted derivative of enhanced green fluorescent protein (EGFP) and can be specifically excited with EGFP filter blocks (Garrels et al, 2012b). Colonies expressing Venus could be detected 3 days after electroporation.…”

Assessment of Fetal Cell Chimerism in Transgenic Pig Lines Generated by Sleeping Beauty Transposition

“…Terminally differentiated gametes of these animals were also Venus positive [32]. A macroscopic inspection of inner organs revealed tissue-specific expression levels of Venus [32].…”

“…Then a transgene specific probe labelled with digoxigenin was used for hybridization as described [29], [31], [32].…”

Characterization of Growth and Reproduction Performance, Transgene Integration, Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer

“…The transposases currently in use for these active transgenesis techniques are piggyBac ( PB ) and Sleeping Beauty ( SB ), which are Class II cut and paste transposase enzymes [14,17,18]. Both PB and SB transposase systems are highly efficient in producing transgenic pigs at a rate around 8% (TAs/micromanipulated embryos), which is much higher than the rates of PNI and ICSI-Tr.…”

“…Both PB and SB transposase systems are highly efficient in producing transgenic pigs at a rate around 8% (TAs/micromanipulated embryos), which is much higher than the rates of PNI and ICSI-Tr. Furthermore, it has been shown that transgenic pigs produced by SB system exhibit normal fecundity, and germline transmission of the transgene [17,19]. …”