Figure 4. Sox2 Reduces Efficiency of NS Cell Conversion to Pluripotency.
(A) Genomic PCR analysis for retroviral transgenes in 2i-iPS clones generated from NS cells that were infected with the four factors.
(B) Southern blot analysis for Sox2, Oct4, Klf4, and Myc retroviral integrations in fNS 2 cells. Arrowheads indicate bands corresponding to retroviral integrations and red dots those of the endogenous genes. Multiple bands in the Sox2 and Oct4 lanes are attributable to cross-hybridisation and pseudogenes, respectively.
(C) Chimera generated from injection of fNS 2 2i-iPS cells into C57BL/6 host blastocyst with C57BL/6 mate and pups. Offspring coat colour demonstrates transmission of an fNS2-derived haploid genome.
(D) Genomic PCR analysis for retroviral segregation in the offspring. Oct4 reporter (GFP), which is homozygous in fNS 2 cells, is used as a positive control. Absence of Oct4 and Klf4 amplification product in some offspring confirms the low transgene copy numbers.
(E–G) Comparison of the reprogramming efficiency of aNS cells infected with either four factors or three factors. 8 × 105 aNS cells were plated and medium switched to 2i five days after infection.
(E) Examples of emerging colonies. Oct4 reporter (GFP) expression indicates pluripotent status.
(F) Colony counts performed at day 8 after 2i switch.
(G) Plates stained for AP 11 d after 2i switch. Puromycin selection was applied 3 d prior to staining. Reprogramming efficiency was calculated taking into account total number of GFP expressing colonies at day 11, 371, and 1137 for four- and three-factors plates, respectively, the number of plated cells, and assuming a 42% probability for a given cell being infected simultaneously with Oct4, Klf4, and c-Myc.
(H) RT-PCR analysis for pluripotency markers in 2i-iPS cells (–Sox2) derived from aNS and fNS of Oct4-GFP mixed background and C57BL/6 inbred backgrounds, respectively. (I and J) Immunofluorescence staining for Oct4 and me3K27 (I) or Nanog (J) in 2i-iPS cells generated without exogenous Sox2.