A method for genetic modification of human embryonic stem cells using electroporation.
The ability to genetically modify human embryonic stem cells (HESCs) will be critical for their widespread use as a tool for understanding fundamental aspects of human biology and pathology and for their development as a platform for pharmaceutical discovery. Here, we describe a method for the genetic modification of HESCs using electroporation, the preferred method for introduction of DNA into cells in which the desired outcome is gene targeting. This report provides methods for cell amplification, electroporation, colony selection and screening. The protocol we describe has been tested on four different HESC lines, and takes approximately 4 weeks from electroporation to PCR screening of G418-resistant clones.
Costa
Magdaline
M
Monash Immunology and Stem Cell Laboratories, STRIP 1, West Ring Road, Monash University Campus, Clayton, Victoria 3800, Australia.
Dottori
Mirella
M
Sourris
Koula
K
Jamshidi
Pegah
P
Hatzistavrou
Tanya
T
Davis
Richard
R
Azzola
Lisa
L
Jackson
Steven
S
Lim
Sue Mei
SM
Pera
Martin
M
Elefanty
Andrew G
AG
Stanley
Edouard G
EG
D002460:Cell Line; D018274:Electroporation / Q000379:methods*; D053595:Embryonic Stem Cells / Q000166:cytology*; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D006801:Humans