UP1 Extends Life of Primary Porcine Fetal Fibroblasts in Culture

Abstract

Genetic modification of somatic cell nuclei and subsequent nuclear transfer has opened an opportunity to create gene-targeted animals. However, somatic cells have a limited life span in culture and it is not possible to introduce precise genetic changes in both alleles in this narrow time window. To increase the life span of somatic cell in culture, both genetic and chemical approaches have been tried with varying success. Here, we report the effect of two anti-oxidants, glutathione and n-t-butyl hydroxylamine, and of the expression of UP1, a shortened derivative of heterogeneous nuclear riboprotein (hnRNP)A1, on the life extension of primary porcine fibroblasts in culture. Under our experimental conditions, the use of anti-oxidants did not result in any prolongation of the life span. In contrast, UP1 expression increased the life span significantly. While most control cells stopped growing by PDL 20, and none survived beyond PDL 35, 100% of UP1-expressing clones reached PDL50, and 40% made it to PDL65. The five UP1-expressing clones were karyotyped at PDL 50. While all of them had a range of numerical chromosomal abnormalities, two clones retained 30-40% normal cells, all the cells in other three clones had abnormal chromosome numbers. Thus, expression of UP1 may be useful in extending the life span of somatic cells in culture. This, in turn, will facilitate the process of gene targeting in this cell type.

Cytolytic assessment of hyperacute rejection and production of nuclear transfer embryos using hCD46-transgenic porcine embryonic germ cells

“…Porcine EG cells may be more amenable to reprogramming after reconstruction than differentiated somatic cells. Furthermore, the chromosomal stability of porcine EG cells, as previously reported (Shim et al, 1997), may yield consistent results in NT compared with somatic cells, such as fetal fibroblasts, that often exhibit chromosomal abnormalities in long-term culture (Mir et al, 2003). Successful acceptance of xenografts occurs after overcoming a series of obstacles, including hyperacute, acute vascular, cellular, and chronic rejection (Khalpey et al, 2004;Yang & Sykes, 2007).…”

“…Porcine EG cells may be more amenable to reprogramming after reconstruction than differentiated somatic cells. Furthermore, the chromosomal stability of porcine EG cells, as previously reported (Shim et al ., 1997), may yield consistent results in NT compared with somatic cells, such as fetal fibroblasts, that often exhibit chromosomal abnormalities in long-term culture (Mir et al ., 2003).…”

Unique proteomic features induced by a potential antiglioma agent, Nordy (dl‐nordihydroguaiaretic acid), in glioma cells

“…Studies showed that the use of antioxidants did not result in any prolongation of the life span, however, Up1 expression increased the life span significantly. Thus, expression of Up1 may be useful in extending the life span of somatic cells in culture [35]. Therefore, Up1 down-regulation found in this study may contribute to the apoptosis as well as the growth inhibition of glioma cells in vitro and in vivo.…”

Increased methylation of the cytosolic 20-kD protein is accompanied by liver regeneration in a hepatectomized rat

“…UP1 has been shown to bind to DNA carrying single-stranded telomeric extensions at the 3' terminus and protects telomeric sequences against degradation by endo-and exonucleases (Dallaire et al, 2000). Furthermore, recent studies have demonstrated that overexpression of UP1 extends life of primary porcine fetal fibroblasts in culture by preventing telomere shortening (Mir et al, 2003). Thus, it is tempting to speculate that UP1 becomes methylated upon proliferating stimuli in a regenerating liver.…”