Eg 400

Immune-deficient F344/NJc1-rnu/rnu female nude rats, six weeks old, were obtained from CLEA Japan. Disposable micropipettes (1-000-0500, Drummon, Alabama, USA) were pulled using a micropipette puller (P-97/IVF Puller, Sutter Instrument, California, USA), and the tip was cut and sharpened using a microgrinder (EG-400, Narishige, Tokyo, Japan). The micropipettes were then adapted to a microelectrode holder (MPH310, World Precision Instruments., FL, USA) on a 6.3 mm electrode handle (2505, World Precision Instruments.), connected to a 10 µl micro-syringe (1701LT, Hamilton, MA, USA) with an extension tube. The animals were anesthetized by inhalation of 5% isoflurane in air, and the eyes were dilated with 0.4% tropicamide. The hiPSC-RPE strip was loaded in a micropipette and the strip was transplanted subretinally.

Caenorhabditis elegans strain N2 and Escherichia coli strain OP50-1 (stm^r) were obtained from the National BioResource Project (NBRP). Caenorhabditis elegans worms were subcultured at 20 °C on NGM agar medium, on which E. coli OP50-1 had been grown. The plasmids were injected into the gonads of young adult worms, and the worms were recovered after injection using well-established methods [23 (link),24 (link)]. The concentration of pCFJ90 was adjusted to 2.5 ng/μL and that of pUC–pro–PonAAS2::GFP–ter to 200, 500, or 1000 ng/μL. The microinjection needles were prepared from borosilicate glass capillaries (GD-1, Narishige, Tokyo, Japan) using a capillary pulling device (PN-30, Narishige). The needle tip was opened with a microforge (MF-900, Narishige), and the opened tip was sharpened using a grinder (EG-400, Narishige). After the plasmid vector was injected, the worms were restored in M9 buffer. A stereomicroscope (SZX-10, Olympus, Tokyo, Japan) equipped with a coaxial epifluorescence system (SZX-RFL2, Olympus) was used to select lines of C. elegans showing fluorescence. F1 individuals showing both mCherry red fluorescence and GFP green fluorescence were selected, and in subsequent generations, those with GFP fluorescence were selected and cultured successively. Photographs of the fluorescent worms were taken using a fluorescence microscope (APEXVIEW APX100, Olympus).