Hanqin Li, Oriol Busquets, Yogendra Verma, Khaja Mohieddin Syed, Nitzan Kutnowski, Gabriella R Pangilinan, Luke A Gilbert, Helen S Bateup, Donald C Rio, Dirk Hockemeyer, Frank Soldner (2022) Highly efficient generation of isogenic pluripotent stem cell models using prime editing eLife 11:e79208
License: This is an open access collection 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 collection and it's working
Created: January 12, 2023
Last Modified: December 13, 2024
Collection Integer ID: 75230
Keywords: ASAPCRN, cultured hpscs for nucleofection, nucleofection nucleofection of hpscs electroporation, cultured hpscs for electroporation, plasmids for nucleofection, free hpscs for nucleofection, hpscs electroporation, plasmids for electroporation, preparing plasmid, mrna for nucleofection, cultured hpsc, preparing mrna, delivery of plasmid, transfected cell, enrichment of transfected cell, human pluripotent stem cell, rnp for nucleofection, using nucleofection, free hpsc, nucleofection nucleofection, hpsc, term hpsc, nucleofection, ribonucleoprotein, plasmid, mrna, described procedure, electroporation, standard procedure, biorad, standard procedure for the delivery
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000486
Abstract
This collection describes the standard procedure for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) into human pluripotent stem cells (hPSCs) using nucleofection or electroporation.
Collection Overview
Preparing MEF-cultured hPSCs for nucleofection
Preparing Feeder-free hPSCs for nucleofection
Preparing plasmids for nucleofection
Preparing mRNA for nucleofection
In vitro assembling of RNP for nucleofection
Nucleofection of hPSCs
Electroporation of hPSCs
A. Preparation of MEFs-cultured hPSCs for electroporation
B. Preparing plasmids for electroporation
C. Electroporation
FACS-based enrichment of transfected cells
A. Preparation of samples for FACS sorting
B. After FACS
General notes
Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
Materials
Item
Vendor
Catalog #
DMEM/F12
Thermo Fisher
11320082
DPBS w/o calcium and magnesium
Corning
MT21031CV
Fetal Bovine Serum (FBS)
Corning
35-011-CV
Knockout Serum Replacement
Thermo Fisher
10828-028
L-Glutamine
Sigma
G8540
Penicillin & Streptomycin
(100X)
Thermo Fisher
15140163
MEM Non-Essential Amino Acids (100X)
Thermo Fisher
11140050
Heat Stable Recombinant Human FGF2
Thermo Fisher
PHG0360
Collagenase type IV
Thermo Fisher
17104019
2-Mercaptoethanol
Sigma
M3148
mTeSR-plus
STEMCELL
Technologies
100-0276
StemFlex
Thermo Fisher
A3349401
Vitronectin (VTN-N) Recombinant Human Protein, Truncated
Thermo Fisher
A14700
Accutase
Thermo Fisher
SCR005
Dispase
STEMCELL
Technologies
NC9995391
Y-27632
Chemdea
CD0141
Cas9, purified protein, 40uM
Macrolab, QB3 UC Berkeley
Synthetic pegRNAs
IDT or Synthego
Synthetic sgRNAs
Synthego
P3 primary Cell 4D X kit S
Lonza
V4XP-3032
Countess™ Cell Counting Chamber Slides
Thermo Fisher
C10228
pCMV-PE2
Addgene
132775
4D-Nucleofector TM Core + X Unit
Lonza
AAF-1002B,
AAF-1002X
5 ml polystyrene round-bottom tube with cell-strainer cap
