Production of a transgenic hPD-L1 pig for xenotransplantation with the Sleeping Beauty transposon system

The Sleeping Beauty (SB) transposon system allows for highly efficient, non- viral integration of new transgenes into the mammalian genome. Here, we report the production of a transgenic piglet carrying a construct for the expression of the human programmed death-ligand 1 (hPD-L1) protein using a SB transposon vector and the hyperactive form of the SB transposase (x100 SB). Due to its ability to provide inhibitory signals to T lymphocytes by engaging the PD-1 receptors on their cell surfaces, the expression of hPD-L1 may contribute to the prevention of the xenograft rejection by the host’s immune system. In order to introduce this gene into porcine cells, we constructed a SB transposon carrying an expression cassette with PD-L1-IRES-GFP under the control of a constitutive CAG promoter. For the purpose of selecting the hPD-L1-positive cells, the transposon vector also contained a doxycycline (DOX)-inducible puromycin- resistance expression cassette. Porcine fetal fibroblasts were transfected by electroporation with the described pSB-TetO-rtTA-ires-Puror-pA-CAG-PD-L1-IRES-GFP-pA transposon together with a plasmid carrying the x100 SB transposase and selected with 5 µg/ml DOX and 2 µg/ml puromycin for 11 days. Analysis of hPD-L1 expression by FACS revealed that 60% of the cells expressed high levels of the transgene. The transgenic fibroblasts were used in two subsequent nuclear transfer experiments and a total of 408 reconstructed embryos were transferred into 4 recipient sows. Two of the recipients became pregnant and one delivered one healthy piglet in addition to one mummified fetus. An ear biopsy from the piglet was used to establish an in vitro culture of dermal fibroblasts, which we used for analysis of hPD-L1 integration and expression. The integration of the expression cassette was confirmed by PCR using genomic DNA as template, while the expression of the PD-L1-ires-GFP construct was demonstrated by GFP fluorescence and RT-PCR. Analysis of blood cells by flow cytometry further confirmed the presence of the human PD-L1 transgene on the surfaces of leucocyte cells, with relatively higher expression being detected in monocytes compared with lymphocytes and neutrophils. In contrast, expression of the puromycin-resistance cassette was not detected, thus fulfilling a secondary objective to avoid the expression of unnecessary antibiotic resistance genes in the transgenic animal. In conclusion, we have successfully produced a piglet expressing hPD-L1 with the use of the SB transposon system.