A method for high efficiency YAC lipofection into murine embryonic stem cells.
We describe a modified protocol for introducing yeast artificial chromosomes (YACs) into murine embryonic stem (ES) cells by lipofection. With a decreased DNA:cell ratio, increased concentration of condensing agents and altered culture conditions, this protocol reduces the requirement for YAC DNA to a few micrograms, improves the recovery of neomycin-resistant ES colonies and increases the yield of clones containing both flanking vector markers and insert. These modifications enable generation of sufficient 'intact' transgenic clones for biological analysis with a single experiment.
Lee
J T
JT
Whitehead Institute for Biomedical Research, Biology Department, Massachusetts Institute of Technology, Cambridge 02142, USA. jlee@wi.mit.edu
Jaenisch
R
R
D000818:Animals; D002478:Cells, Cultured; D018244:Chromosomes, Artificial, Yeast; D002999:Clone Cells; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D013234:Stem Cells / Q000378:metabolism*; D014162:Transfection / Q000379:methods*