Descrizione: The derivation of induced pluripotential cell (iPSC) lines from somatic cells undergoing Whole Genome Sequencing (WGS) provides a complementary resource for conservation and compar-ative genomic studies. Routine reprogramming of human patient cells is being used to capture the genetic diversity of individuals included in Genome Wide Association Studies (GWAS) to generate new cell models that can recapitulate developmental and disease processes in a dish. This strategy can be extended across genera to generate unlimited amounts of pluripotent cells from individual animals included in WGS projects. The conservation of identity of key transcription factors is sufficient that transduction with pseudo¬typed retroviral vectors expressing the human Oct4, Sox2, Klf4 and c-Myc cDNAs can reprogram somatic cells from a wide range of mammals without the need to clone species specific genes. One concern of this retroviral approach is that the putative iPSC lines can become transformed, due to incomplete silencing of the integrated transgenic proto-oncogenes preventing differentiation, as well as promoting the accumulation of aneuploidies. To overcome these technical hurdles, non-integrating reprogramming methods including transduction with mRNA viral vectors and daily transfection with synthetic mRNAs were compared and have proven effective in reprogramming fibroblasts from a range of canine, feline and Non-Human Primate species. Of practical utility, these putative iPSC lines grow rapidly and can be cultured for over 15 passages, well beyond the point at which the parental fibroblast lines undergo senescence. The majority of putative iPSC lines derived by these non-integrating methods maintain a normal diploid karyotype by Giemsa banding. Standard in vitro and in vivo assays are underway to test the plurip¬otency of these cell lines. In addition, WGS is being performed on select samples to compare the genomes before and after repro¬gramming to make sure that no significant genetic drift has occurred in culture. RNA-based reprogramming provides a reproducible approach to generating viable cell models free of DNA-artifacts and can add value to current genome resource banking efforts.
Non-integrating Reprogramming of Mammalian Cells for Genome Resource Banking
Reinbold R;Zucchi I;
2017
Abstract
Descrizione: The derivation of induced pluripotential cell (iPSC) lines from somatic cells undergoing Whole Genome Sequencing (WGS) provides a complementary resource for conservation and compar-ative genomic studies. Routine reprogramming of human patient cells is being used to capture the genetic diversity of individuals included in Genome Wide Association Studies (GWAS) to generate new cell models that can recapitulate developmental and disease processes in a dish. This strategy can be extended across genera to generate unlimited amounts of pluripotent cells from individual animals included in WGS projects. The conservation of identity of key transcription factors is sufficient that transduction with pseudo¬typed retroviral vectors expressing the human Oct4, Sox2, Klf4 and c-Myc cDNAs can reprogram somatic cells from a wide range of mammals without the need to clone species specific genes. One concern of this retroviral approach is that the putative iPSC lines can become transformed, due to incomplete silencing of the integrated transgenic proto-oncogenes preventing differentiation, as well as promoting the accumulation of aneuploidies. To overcome these technical hurdles, non-integrating reprogramming methods including transduction with mRNA viral vectors and daily transfection with synthetic mRNAs were compared and have proven effective in reprogramming fibroblasts from a range of canine, feline and Non-Human Primate species. Of practical utility, these putative iPSC lines grow rapidly and can be cultured for over 15 passages, well beyond the point at which the parental fibroblast lines undergo senescence. The majority of putative iPSC lines derived by these non-integrating methods maintain a normal diploid karyotype by Giemsa banding. Standard in vitro and in vivo assays are underway to test the plurip¬otency of these cell lines. In addition, WGS is being performed on select samples to compare the genomes before and after repro¬gramming to make sure that no significant genetic drift has occurred in culture. RNA-based reprogramming provides a reproducible approach to generating viable cell models free of DNA-artifacts and can add value to current genome resource banking efforts.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
