Journal: Stem Cell Reports
Article Title: NANOG Is Required for the Long-Term Establishment of Avian Somatic Reprogrammed Cells
Figure Lengend Snippet: Attempts to Obtain Chicken Reprogrammed Cells with Various Delivery Systems Primary chicken embryonic fibroblasts (CEFs) were infected with the pLent polycistronic lentivirus. Colonies emerged (A) and expressed GFP (B), indicating that they were infected, although they failed to be amplified. More than 50 colonies were isolated in several independent experiments. Similarly, CEFs were infected with a cocktail of individual lentiviruses carrying mouse Oct4 , Sox2 , Klf4 , and c-Myc cDNAs. Compact colonies with AP-positive cells were obtained (C and D) but could not be established long term. In another attempt, CEFs were infected with pMX retroviruses carrying mouse Oct4 , Sox2 , Klf4 , and c-Myc . Morphological changes were rapidly observed (E), and colonies expanded once individually picked (F). The phenotypic changes were not stable, and cells reverted to a fibroblast phenotype (G) after four to six passages. Similarly, CEFs infected with Sendai viruses carrying human OCT4 , SOX2 , KLF4 , and c-MYC cDNAs exhibited morphological changes (H) but failed to be established and reverted to a fibroblast phenotype after a few passages. Scale bars, 50 μm.
Article Snippet: The cDNAs from chicken POUV ( POU5F3 ), SOX2 , SOX3 , KLF4 , c-MYC , NANOG , and LIN28 were cloned, sequenced, inserted into the pGAE lentivirus or pPB transposon backbones, and deposited to Addgene.
Techniques: Infection, Amplification, Isolation