Characteristics of primary and immortalized fibroblast cells derived from the miniature and domestic pigs

Oh, Ho Yeon; Jin, Xun; Kim, Jong Geun; Oh, Myung Joo; Pian, Xumin; Kim, Jun Mo; Yoon, Moon Seok; Son, Chae Ik; Lee, Young Sik; Hong, Kyung Pyo; Kim, Hyunggee; Choi, Yun Jaie; Whang, Kwang Youn

doi:10.1186/1471-2121-8-20

Cited by 41 publications

(33 citation statements)

References 56 publications

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“…Immortalization of porcine fetal fibroblasts by using hTERT alone was shown by Moon et al, (2014). However, other groups were not able to achieve the same results using fibroblasts from miniature or domestic pigs (Oh et al, 2007).…”

Section: Immortalization Of Cells Using Htertmentioning

confidence: 93%

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Multi‐transgenic pigs for xenotransplantation

Fischer

Kraner-Scheiber

Flisikowski

et al. 2013

Xenotransplantation

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Genetically modified pigs offer a solution to the severe shortage of organs available for human transplantation. Inactivation of the α1,3‐galactosyltransferase gene, responsible for α1,3‐Gal epitope synthesis (1–2) was a major breakthrough and essentially solved the problem of hyperacute rejection. However, further modifications of donor pigs are required to enable long term xenograft survival. These include expression of complement regulatory, antithrombotic, endothelium protective and immuno‐regulatory transgenes. Current evidence indicates that complement regulator transgenes, such as CD46 and CD55, must be expressed at least fivefold more than their human endogenous equivalent to effectively protect grafted tissue (3). To explore means of achieving high expression, we produced a series of BAC‐ and PAC‐vector constructs containing different sized genomic complement regulatory genes, from 70 to 190 kb, under the control of endogenous or CAGGs (CMV enhancer chicken β actin) promoter sequences. Using these vectors we obtained high levels of CD46 and CD55 expression in vitro. To prepare multi‐transgene “packages” for introduction into animals, the most effective constructs were combined with one or two further xenoprotective transgenes, including A20 (4), HO‐1 (5), thrombomodulin (6) and CTLA4‐Ig (LEA 29Y). In cotransfection experiments single cell clones expressing up to five different xenogenes could be detected. Achieving desired levels of transgene expression in an animal is not only a function of the transgene construct, but also of the location at which it integrates. Position effect variation in expression of random transgenes is well‐documented. Transgene placement by gene targeting at a permissive locus offers a useful means of achieving reliable transgene expression. In mice the ROSA26 locus is widely used to support ubiquitous expression of inserted transgenes (7–8). Murine ROSA26 encodes no functional genes and extensive gene targeting of this locus has not led to deleterious effects on development or physiology (9). To enable a similar approach in pigs, we recently identified a strong candidate for the porcine ROSA26 locus. Gene targeted placement of a neomycin reporter gene construct under the control of the ROSA26 promoter showed expression in all examined porcine tissues of all three germ layers. This strongly suggests that porcine ROSA 26 may resemble the murine locus as a suitably permissive site for transgene expression. We are currently proceeding with gene targeting to place various xenoprotective transgene constructs at this locus. References: 1. Dai Y, Vaught TD, Boone J et al. Targeted disruption of the alpha1,3‐ galactosyltransferase gene in cloned pigs. Nat Biotechnol 2002; 20: 251–255. 2. Lai L, Kolber‐Simonds D, Park K et al. Production of alpha‐1,3‐galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002; 295: 1089–1092. 3. Miyagawa S, Fukuta D, Kitano E et al. Effect of tandem forms of DAF(CD55) on complement‐mediated xenogeneic cell lysis. Xenotransplantation 20...

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Section: Immortalization Of Cells Using Htertmentioning

confidence: 93%

“…Porcine hepatocyte lines were immortalized by the use of SV40LT and hTERT (Pan et al, 2008) which worked also efficient for porcine fibroblasts (Oh et al, 2007). Immortalization of porcine fetal fibroblasts by using hTERT alone was shown by Moon et al, (2014).…”

Section: Immortalization Of Cells Using Htertmentioning

confidence: 99%

Multi‐transgenic pigs for xenotransplantation

Fischer

Kraner-Scheiber

Flisikowski

et al. 2013

Xenotransplantation

View full text Add to dashboard Cite

show abstract

“…7) In brief, the continuous expression of SV40T for more than 60 passages increased the level of polyploidy to about 60%. 10) We analyzed the karyotype of SV40-expressing PEF at passage 10 in our study and found that the level of polyploidy was 16.4% (21/128 samples, see Table 1). In the case of the wild-type PEF cells, the level of polyploidy was about 6.5%.…”

Section: Discussionmentioning

confidence: 99%

“…This result is consistent with the previous findings. 10) Although the expression of SV40T was quite effective for establishing cell lines, prolonged expression has been reported to cause chromosomal abnormalities. 7) In brief, the continuous expression of SV40T for more than 60 passages increased the level of polyploidy to about 60%.…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies have reported that the expression of SV40T over longer periods induced karyotype abnormalities. 7,10) We analyzed the karyotype of 107 wild-type PEF for compar-A C B ison. Although more than 90% of wild-type PEF showed the normal karyotype, 6.5% of wild-type PEF were 4n, possibly because of a side effect of the colcemid treatment for karyotyping.…”

Section: Methodsmentioning

confidence: 99%

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