A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis.
The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take approximately 1 month.
Davis
Richard P
RP
Monash Immunology and Stem Cell Laboratories, STRIP 1, Building 75, Level 3, Monash University, Clayton, Victoria, Australia.
Costa
Magdaline
M
Grandela
Catarina
C
Holland
Andrew M
AM
Hatzistavrou
Tanya
T
Micallef
Suzanne J
SJ
Li
Xueling
X
Goulburn
Adam L
AL
Azzola
Lisa
L
Elefanty
Andrew G
AG
Stanley
Edouard G
EG
D004352:Drug Resistance, Microbial / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016254:Mutagenesis, Insertional / Q000379:methods*