Fig 1.
Schematic outline of reprogramming and programming to lens differentiation.
The founder mouse was generated by in ovo transposition. Fibroblasts were isolated from a cryTom positive fetus at d11.5. At passage 2, the fibroblasts were reprogrammed to iPS cells by co-electroporation of a Sleeping Beauty reprogramming transposon and a SB expression plasmid as previously described [32, 49, 50]. The iPS cells were seeded on mitotically inactivated cell feeders (NTERA and P19), hypothesizing that the feeder will provide a niche for ectoderm and lens cell differentiation. Differentiation into lens cells should result in re-activation of the Cryaa-tdTomato reporter.
Fig 2.
Exclusive expression in eye lens during fetal development.
(A) Murine fetus at day 10.5 p.c., A´) corresponding fluorescence image. (B) Murine fetus at day 11.5 p.c., B´) corresponding fluorescence image. (C) Murine fetus at day 12.5 p.c., C´) corresponding fluorescence image, note the onset of tdTomato expression in the forming lens area (arrow). (D) Higher magnification of the d12.5 fetus, overlay, D´) brightfield and D”) fluorescence images. (E) Murine fetus at day 13.5 p.c., E´) corresponding fluorescence image. (F) Murine fetus at day 14.5 p.c., F´) corresponding fluorescence image. Size bars = 1 mm.
Fig 3.
TdTomato expression in the adult eye.
(A) TdTomato expression in the isolated mouse eye, A´) corresponding brightfield view, size bar = 1 mm. (B) TdTomato expression in ciliary muscle, B´) corresponding brightfield view, note the drastically increased exposure time relative to the lens to reveal expression in muscle, Size bar = 1 mm. (C) Immunoblot detection of tdTomato during prenatal stages, the full-sized tdTomato of about 54 kDa is detected (black arrow). In the adult lens several smaller degradation products are found (red arrows). M, molecular weight marker; cryTom, samples from transgenic animals and fetuses; wt, wildtype controls. (D) Western blotting of tubulin (loading control). (E) Expression of endogenous alphaA crystallin is similar in transgenic and wildtype animals. Top, Western blotting of tdTomato; bottom, Western blotting of endogenous alphaA crystallin with a polyclonal antibody; bottom, Coomassie stained gels as loading controls. M, molecular size marker; 1, eye lens; 2, ZNS; 3, cerebellum; 4, lung; 5, heart; 6, skel. muscle; 7, kidney; 8, skin; and 9, liver.
Fig 4.
Reduced light transmission in tdTomato expressing lenses.
(A) Isolated lenses from a cryTom and a wildtype animal are transmitted with white light from below, and grey scale images are recorded. The dotted lines indicate the measurement areas (see C), size bar = 1 mm. (B) Corresponding fluorescence image. (C) Quantification of light transmittance. Note that the wildtype lens (black line) shows almost complete light transmittance, whereas the cryTom lens (red line) shows a reduced transmittance. Measurement areas are the dotted lines indicated in A).
Fig 5.
Characterization of murine iPS cells.
(A) Schedule for non-viral iPS cell generation by SB transposon reprogramming [32, 49, 50]. (B) Initial colonies formed 9–15 days post electroporation. Bar = 20 micrometer. (C) AP stained culture 15 days post electroporation. Note, the intensively red stained colonies. Bar = 20 micrometer. (D) Upon culture in hanging drops, embryoid bodies formed readily. Bar = 50 micrometer. (E) Upregulation of stemness-related genes in the cryTom iPS cells.
Fig 6.
Characterization of in vitro-formed lentoid bodies.
(A) Lentoid bodies with tdTomato expression derived from a co-culture of cryTom iPS on NTERA-2, A´) corresponding brightfield view. Bar = 50 micrometer. (B) Individual tdTomato-positive cells derived from a co-culture of cryTom iPS on P19, B´) overlay; B´´) corresponding brightfield view. Bar = 50 micrometer. (C) Lentoid body with tdTomato expression derived from a co-culture of cryTom iPS on P19, C´) overlay; C´´) corresponding brightfield view. Bar = 50 micrometer. (D) Expression analyses of co-cultures by RT-PCR. The endogenous murine Cryaa gene could be detected in P19 co-cultures. The endogenous lens-specific CryF transcript could be detected in NTERA-2/iPS and P19/iPS co-cultures, but also in P19 cells. CoN, co-culture of NTERA-2 and iPS; CoP19, co-culture of P19 and iPS; N, NTERA-2; P19, P19 cells; eye, positive control; -RT, without reverse transcriptase; H20, no template. (E) Immunodetection of tdTomato protein. CoN, co-culture of NTERA-2 and iPS; CoP19, co-culture of P19 and iPS; N, NTERA-2; P19, P19 cells. (F) Expression analysis of co-cultures for key regulatory genes, Pax6 and Prox1, of lens differentiation by RT-PCR. CoN, co-culture of NTERA-2 and iPS; CoP19, co-culture of P19 and iPS; F, fibroblasts.