Mechanism of prolonged gene expression by Epstein–Barr virus‐based plasmid in porcine cells

Son, Jung Kyu; Oh, Sang Taek; Cho, Sung Kyu; Yoon, Kun Ho; Lee, Suk Kyeong

doi:10.1111/j.1399-3089.2006.00350.x

Cited by 4 publications

(3 citation statements)

References 22 publications

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“…In previous studies, Lipofectamine-mediated transfection of swine kidney epithelial cells with episomal vectors gave a 20e30% transfection yield [27], and FuGENE 6-mediated transfection of human embryonic stem cells with episomal vectors gave a 30e40% transfection yield [10]. In both of these cases, an immortalized cell line was used.…”

Section: Discussionmentioning

confidence: 99%

“…While this method was successful in cancerous cell lines [10,27], there are no reports of successful delivery into primary cells obtained from fresh tissue. Therefore, in this study we have identified some potential commercial transfection reagentsdwhich are inexpensive, involve simple handling, and show low toxicity and immunogenicitydfor transport of episomal vectors into primary cells, and we evaluated their cytotoxicity and transfection efficiency.…”

Section: Introductionmentioning

confidence: 89%

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Delivery of episomal vectors into primary cells by means of commercial transfection reagents

Han

Lee

Baek

et al. 2015

Biochemical and Biophysical Research Communications

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Although episomal vectors are commonly transported into cells by electroporation, a number of electroporation-derived problems have led to the search for alternative transfection protocols, such as the use of transfection reagents, which are inexpensive and easy to handle. Polyplex-mediated transport of episomal vectors into the cytoplasm has been conducted successfully in immortalized cell lines, but no report exists of successful transfection of primary cells using this method. Accordingly, we sought to optimize the conditions for polyplex-mediated transfection for effective delivery of episomal vectors into the cytoplasm of primary mouse embryonic fibroblasts. Episomal vectors were complexed with the commercially available transfection reagents Lipofectamine 2000, FuGEND HD and jetPEI. The ratio of transfection reagent to episomal vectors was varied, and the subsequent transfection efficiency and cytotoxicity of the complexes were analyzed using flow cytometry and trypan blue exclusion assay, respectively. No cytotoxicity and the highest transfection yield were observed when the ratio of transfection reagent to episomal vector was 4 (v/wt) in the cases of Lipofectamine 2000 and FuGENE HD, and 2 in the case of jetPEI. Of the three transfection reagents tested, jetPEI showed the highest transfection efficiency without any cytotoxicity. Thus, we confirmed that the transfection reagent jetPEI could be used to effectively deliver episomal vectors into primary cells without electroporation.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 89%

Delivery of episomal vectors into primary cells by means of commercial transfection reagents

Han

Lee

Baek

et al. 2015

Biochemical and Biophysical Research Communications

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“…Son et al. [22] investigated the mechanisms underlying the prolonged gene expression of Epstein–Barr virus (EBV)‐based plasmids, pEBV‐GFP, in porcine neonatal pancreatic cell clusters. They observed that the EBV‐based plasmids, pEBV‐GFP, remained as episome in transfected porcine cell clusters, rather than integrating into the chromosomal DNA.…”

Section: Gene Therapymentioning

confidence: 99%

Xenotransplantation literature update November–December 2006

Baertschiger¹,

Bühler²

2007

Xenotransplantation

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Induction of Efficient Differentiation and Survival of Porcine Neonatal Pancreatic Cell Clusters Using an EBV-based Plasmid Expressing HGF

Kim

Sun

et al. 2007

Journal of Biochemistry

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Porcine neonatal pancreatic cell clusters (NPCCs) have been actively studied as a source of pancreatic stem cell transplantation for the treatment of diabetes. In this study, the hepatocyte growth factor (HGF) gene was cloned in an Epstein-Barr virus (EBV)-based plasmid vector (pEBVHGF) and the effects of the HGF expression on the survival and differentiation of NPCCs were analysed. For comparison, pHGF was constructed by deleting EBNA-1 and OriP from pEBVHGF. The expression of HGF, as measured by ELISA, lasted longer when pEBVHGF was used than when pHGF was used. C-Met phosphorylation co-related with the expression of HGF in the transfected NPCCs. Immunocytochemistry experiments showed that NPCCs showed a higher and longer expression of insulin when they were transfected with pEBVHGF than with pHGF. Moreover, a greater number of NPCCs survived for a longer period after they were transfected with pEBVHGF than when they were transfected with pHGF. Taken together, these results indicate that transfecting NPCCs with the HGF gene using an EBV-based plasmid is a more effective method of inducing differentiation to beta cells and enhancing survival than using a conventional plasmid. Therefore, it may be possible to use EBV-based plasmids to modify pancreatic stem cells for xenotransplantation.

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Mechanism of prolonged gene expression by Epstein–Barr virus‐based plasmid in porcine cells

Abstract: The EBV-based plasmid may be useful for genetically manipulating porcine cells to enhance their value as xenotransplantation sources.

Cited by 4 publications

References 22 publications

Delivery of episomal vectors into primary cells by means of commercial transfection reagents

Delivery of episomal vectors into primary cells by means of commercial transfection reagents

Xenotransplantation literature update November–December 2006

Induction of Efficient Differentiation and Survival of Porcine Neonatal Pancreatic Cell Clusters Using an EBV-based Plasmid Expressing HGF

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