UP1 extends life of primary porcine fetal fibroblasts in culture.
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.
Mir
Bashir
B
Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina.
Tanner
Natalie
N
Chowdhary
Bhanu P
BP
Piedrahita
Jorge A
JA
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D002869:Chromosome Aberrations; D003469:Culture; D018274:Electroporation; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics / Q000378:metabolism*; D006898:Hydroxylamines / Q000494:pharmacology; D007621:Karyotyping; D013552:Swine / Q000196:embryology*
D000975:Antioxidants; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D006898:Hydroxylamines; D005978:Glutathione