Codon optimized membrane cofactor protein expression in α 1, 3 galactosyltransferase knockout pig cells improve protection against cytotoxicity of monkey serum

Lee, Heasun; Hwang, In-Sul; Vasamsetti, Bala Murali Krishna; Rallabandi, Harikrishna Reddy; Park, Mi-Ryung; Byun, Sung June; Yang, Hyeon; Ock, Sun‐A; Lee, Hwi-Cheul; Woo, Jonghye; Hwang, Seongsoo; Oh, Keon Bong

doi:10.1007/s13205-020-2091-z

Cited by 3 publications

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“…PEFs and PAECs were isolated from one-month-old α1,3-galactosyltransferase knockout (GTKO) pigs and cultured as previously described [ 17 ]. PK-15 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen) supplemented with 10% ( v/v ) fetal bovine serum (FBS) and 1× Antibiotic-Antimycotic (Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

“…PK-15 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen) supplemented with 10% ( v/v ) fetal bovine serum (FBS) and 1× Antibiotic-Antimycotic (Thermo Fisher Scientific). The methods for transfecting the PEFs and PAECs were described previously [ 17 ]. The PK-15 cells were transfected using a Lipofectamine 3000 Kit (Invitrogen) according to manufacturer’s protocol.…”

Section: Methodsmentioning

confidence: 99%

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Select Porcine Elongation Factor 1α Sequences Mediate Stable High-Level and Upregulated Expression of Heterologous Genes in Porcine Cells in Response to Primate Serum

Sun

Yang

et al. 2021

Genes

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Genetically engineered (GE) pigs with various combinations of genetic profiles have been developed using heterologous promoters. This study aimed to identify autologous promoters for high and ubiquitous expression of xenotransplantation relevant genes in GE pigs. A 1.4 kb upstream regulatory sequence of porcine elongation factor 1α (pEF1α) gene was selected and isolated for use as a promoter. Activity of the pEF1α promoter was subsequently compared with that of the cytomegalovirus (CMV) promoter, CMV enhancer/chicken β-actin (CAG) promoter, and human EF1α (hEF1α) promoter in different types of pig-derived cells. Comparative analysis of luciferase and mutant human leukocyte antigen class E-F2A-β-2 microglobulin (HLA-E) expression driven by pEF1α, CMV, CAG, and hEF1α promoters revealed the pEF1α promoter mediated comparable expression levels with those of the CAG promoter in porcine ear skin fibroblasts (PEFs) and porcine kidney-15 (PK-15) cells, but lower than those of the CAG promoter in porcine aortic endothelial cells (PAECs). The pEF1α promoter provided long-term stable HLA-E expression in PEFs, but the CAG promoter failed to sustain those levels of expression. For xenogeneic serum-induced cytotoxicity assays, the cells were cultured for several hours in growth medium supplemented with primate serum. Notably, the pEF1α promoter induced significant increases in luciferase and HLA-E expression in response to primate serum in PAECs compared with those driven by the CAG promoter, suggesting the pEF1α promoter could regulate temporal expression of heterologous genes under xenogeneic-cytotoxic conditions. These results suggest the pEF1α promoter may be valuable for development of GE pigs spatiotemporally and stably expressing immunomodulatory genes for xenotransplantation.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%