Streptolysin-O Treatment of Fetal Fibroblasts Improves Cell Fusion and In Vitro Development of Porcine Nuclear Transfer Embryos

Abstract

Abstract. Streptolysin O (SLO) is a known bacterial protein that forms very large pores in the plasma membranes of mammalian cells. SLO has been used in the delivery of proteins into living cells following permeabilization. The objective of this study was to investigate the effects of SLO treatment in donor cells that generate pores in the plasma membrane during in vitro development of porcine reconstructed embryos. In experiment 1, fetal fibroblast cells were trypsinized, treated with SLO for 0, 30, 50 and 70 min and then their fusion rates and developmental capabilities were tested following reconstruction. SLO treatment for 50 min produced a higher blastocyst formation rate compared with the other treatments. In experiment 2, fetal fibroblasts were treated with 200 ng/ml SLO for 50 min at the confluent or single-cell stages of the nuclear transfer protocol (i.e., before and after trypsinization, respectively), and the in vitro development of the resulting porcine reconstructed embryos was investigated over 6 days in culture. Oocytes receiving cells treated with SLO post-trypsinization showed higher fusion and blastocyst formation rates compared with those receiving untreated cells. Collectively, these findings show that SLO-mediated permeabilization of porcine fetal fibroblast cells appears to improve the fusion rates and in vitro development of porcine reconstructed embryos.

Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with Xenopus egg extract and/or digitonin

“…Interestingly, a similar effect on cloning efficiency was observed when at least certain donor cells were pre-treated with digitonin alone. Other authors have demonstrated increased in vitro cloning efficiency after SLO permeabilization of fetal fibroblasts when employed without membrane re-sealing, which correlates with an increased fusion rate of the donor cell and enucleated oocytes during the cloning procedure (Naruse et al ., 2009). In our work, no differences in fusion rates were observed (data not shown), so this finding is most probably not the reason for our observed increase in cloning efficiency.…”

“…SLO and digitonin are the most frequently used agents for extract treatment and cause pores to form in the plasma membrane of sizes that are sufficient for passive diffusion of proteins up to 100 kDa (Adam et al, 1992;Walev et al, 2001). Interestingly, it has also been shown that permeabilization agent without further extract treatment is able to improve the cell fusion and in vitro development of SCNT embryos (Naruse et al, 2009). In contrast, Miyamoto et al (2007b) found that permeabilization and extract treatment could not increase the blastocyst rate; only the cell number of the individual embryos increased.…”

Quantitative analysis of mitochondrial DNA in porcine-mouse cloned embryos

“…Cell culture and assessment were performed as previously described [12]. Porcine fetal fibroblasts (PFF) and mouse fetal fibroblasts (MFF) were used as donor cells for intraspecies and interspecies nuclear transfer, respectively.…”

Pectate Lyase Promoting Streptolysin O Expression in Escherichia coli and Strengthening Its Activity

“…With SLO, 3 active domains of large clostridial toxins (Clostridium difficile B-toxin, C. sordelli lethal toxin, and C. botulinum C2 toxin) were introduced into 3 different cell lines, and the results revealed that all the 3 toxins disrupted the actin cytoskeleton to cause the rounding up of the cells (7). A past study indicated that SLO treatment improved the cell fusion of fetal fibroblasts and the nucleic transferring of porcine embryo (12). Streptolysin O was used as an adjunct therapy with antibiotics to reduce the duration and severity of disease caused by Rhodococcus equipneumonia compared to antibiotic therapy alone (13).…”