Porcine Knock-in Fibroblasts Expressing hDAF on α-1,3-Galactosyltransferase (GGTA1) Gene Locus

Abstract

The Galactose-α1,3-galactose (α1,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of α1,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.

Generation of Fibroblasts Lacking the Sal-like 1 Gene by Using Transcription Activator-like Effector Nuclease-mediated Homologous Recombination

“…For electroporation, the fibroblasts were cultured in DMEM supplemented with 15% defined fetal bovine serum (FBS), 1× non-essential amino acids, 1× sodium pyruvate, 10 −4 M β-mercaptoethanol, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO 2 . Transfection was performed according to a previously reported method ( Kim et al, 2012 ). Briefly, the fibroblasts were harvested by treatment with 0.25% trypsin and were resuspended in Ham’s F10 medium (HyClone Co.; Logan, UT, USA) at a density of 5×10 6 cells per 0.4 mL of the medium.…”

Knock-in of Enhanced Green Fluorescent Protein or/and Human <i>Fibroblast Growth Factor 2</i> Gene into <i>β-Casein</i> Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein

“…The cells were cultured with growth medium (DMEM, 15% FBS, 1×non-essential amino acids, 1×sodium pyruvate, 10 −4 M β-mercaptoethanol, 100 unit/mL penicillin, 100 μg/mL streptomycin) for electroporation. The transfection was conducted according to a previously reported method ( Kim et al, 2012 ), as follows. The cells were trypsinized and resuspended at a concentration of 1.25×10 7 cells/mL in F10 nutrient mixture.…”

“…Preparation of genome DNA from G418-resistant colonies was performed according to a previously reported method ( Kim et al, 2012 ). When the cells were sub-cultured from 24-well plates to 12-well plates, 200 μL of cell suspension from the 24-well plates were recovered by centrifugation and suspension with 40 μL of deionized water containing 0.05 mg/mL proteinase K (Roche, Foster City, CA, USA).…”

The Effect of Long Time of Storage on Physical Quality, Nutrient Content, and In Vitro Digestibility of Cattle Complete Feed

“…These results are in accordance with reports by Jin et al [28] and Chang et al [29], wherein there was a difference in feed intake between TMR-based feeding and SCF during the growing period; however, there was no significant difference in body weight gain. Kim et al [30] argued that compared with SCF feeding, feeding TMR or TMR with fermented feed during the growing period increased daily weight gain, as nutrient use efficiency was improved with fermentation in the rumen.…”