Apr 03, 2023
Generation of induced pluripotent stem cells and gene correction
- 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Protocol Citation: michela.deleidi 2023. Generation of induced pluripotent stem cells and gene correction. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8jo89g2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 01, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 79876
Keywords: ASAPCRN
Abstract
iPSC generation and gene correction (CRISPR-CAS9) protocol
Generation of induced pluripotent stem cells and gene correction
Generation of induced pluripotent stem cells and gene correction
Skin fibroblasts were reprogrammed by nucleofection with pCXLE- hOct3/4 (RRID:Addgene_27076), pCXLE-hSK (RRID:Addgene_27078),using the Amaxa nucleofection kit for human dermal fibroblasts (Lonza, VPD-100) and program P-022 of the Nucleofector 2b (Lonza).
Nucleofected fibroblasts were plated in six-well plates coated with Matrigel (Corning)
in DMEM supplemented with 10% FBS (Gibco) and 1% GlutaMAX Supplement (Gibco).
The following day, the medium was changed to DMEM+/+ (DMEM with 10% FBS and 1% GlutaMAX Supplement and 1% Pen/Strep (Millipore)) supplemented with 2 ng/ml recombinant
basic human fibroblast growth factor (FGF2, Peprotech).
On day 3 or4 post nucleofection, the medium was changed to E8 medium com-
posed of DMEM F12 with HEPES (Gibco), 128 ng/ml ascorbic acid (Sigma
–Aldrich), 1x insulin-transferrin-selenium (Thermo Fisher
Scientific), 10 ng/mL FGF2 (Peprotech), 500 ng/ml heparin (Sigma-Aldrich), and 2 ng/ml TGFβ1 (Peprotech). E8 medium was supplemented with 100 μm sodium butyrate and 0.1% Pen/Strep.
Colonies started to appear from day 14 onward.
Induced pluripotent stem cells (iPSCs) were cultured on Vitronectin XF (StemCell Technologies) in E8 medium.
Gene correction for the L444P mutation was performed as previously described in (Schöndorf, D. C. et al., 2014)
One hour before nucleofection, 10μM Rockinhibitor was added to the iPSC medium. About 240 nM crRNA (IDT): Atto550-labeled tracrRNA (IDT) duplex was complexed with 124μM Cas9 to form the ribonucleoprotein complex (RNP complex).
iPSCs(1.6 × 10^6) were nucleofected with the RNP complex and 16μg of ssODN using 100μl of Ingenio nucleofection solution (Mirus) with program B-016 of Nucleofector 2b.
Following nucleofection, the cells were FACSsorted for Atto550-positive cells using a FACS Aria II with a 100-μmnozzle. After sorting, 1 × 10^4 cells were plated per 10-cm dish.
Colonies were picked and sequenced by Sanger sequencing.