Biomedical Engineering Reference
In-Depth Information
7 Wash three times with ice-cold water as described in steps 3-5 of Protocol 10.2.
8 Resuspend the cell pellet in 50
μ
l of ice-cold water.
9 Add 300 ng of the PCR product obtained from Protocol 10.3 and electroporate as
described in Protocol 10.2, steps 3-11.
10 Plate the electroporated bacteria on LB-agar plates with antibiotic selection for the
plasmid (not the BAC) and incubate overnight at 32
◦
C.
11 Screen colonies by standard
Taq
PCR for the retrieved DNA sequence.
10.2.4 ES and MEF cell culture
The R1 line of ES cells is a well-established line with an impressive track record of germline
transmission of targeted genes. The R1 cell line was derived from deliberate outcrossing of
129/
Sv
and 129/
SvJ
strains. The majority of available ES cell lines were derived from 129
substrains, due to the ability of this strain to generate ES cells that efficiently contribute
to the mouse germline after extensive manipulation in culture. This cell line is available
to the scientific community from the laboratory of Andras Nagy at Mount Sinai hospital
(http://www.mshri.on.ca/nagy/) and in our hands has proven a reliable resource for the
generation of knockout mice. The following protocols have been specifically adapted for
R1 ES cells; culture conditions for other ES cell lines have been successfully developed in
many laboratories.
10.2.4.1 MEF cell culture protocols
ES cells must be maintained in an undifferentiated, healthy status throughout the entire
gene targeting procedure. When using R1 ES cells, one critical aspect is to culture them
on top of a layer of feeder MEF cells. Although it is possible to culture R1 cells without
this feeder layer, it is highly recommended to use MEF cells when possible (ref Nagy web-
site). Protocols 10.5
10.10 describe how to produce MEF cells from a transgenic mouse
line with a neomycin resistance cassette. This cassette will confer resistance to the antibi-
otic G418, which is necessary for positive ES cell clone selection. Additionally, after the
MEF cells are isolated and expanded, they are
γ
-irradiated in order to prevent cell divi-
sion of the feeder cells. One transgenic mouse line expressing the neomycin resistance
cassette, C57BL/6J-Tg(pPGKneobpA)3Ems/J, is commercially available from The Jackson
Laboratory.
−
PROTOCOL 10.5
Isolating MEF Cells (from 8 to 12 embryos)
Equipment and reagents
•
Incubator at 37
◦
Cwith5%CO
2
•
Water bath at 37
◦
C
