Biomedical Engineering Reference
In-Depth Information
There are two aspects that require particular attention when performing RMCE. First, high
levels of Cre-recombinase gene expression are needed. For this, the use of Cre-GFP fusion
vectors allows isolation of GFP-positive cells by cell sorting, increasing the probability of
isolating clones in which the cassette exchange has occurred. Second, the enrichment of
cell population is required in which the cassette exchange takes place. These goals can be
attained by taking advantage of GFP as a reporter gene (whenever possible) to perform
one or even two rounds of cell sorting. This is feasible owing to the fact that positive
reporter-expressing cells will be favored and this particular cell population will be enriched.
For this reason, alternative selection methods are recommended (see Section 7.3).
In conclusion, the RMCE protocol is very efficient, enabling chromatin- and function-
related studies with reproducible results for the analysis of regulatory elements and genera-
tion of varied types of transgenes in different cell lineages, including ES cells [30, 31, 34].
7.2.2 Plasmid requirement
1Weusedthe
CMV-HYTK
gene that can be positively selected by hygromycin and neg-
atively by gancyclovir. A plasmid (pL1-HYTK-L1) containing the
CMV-HYTK
gene
flanked by inverted
loxP
sites is stably transfected and selected with hygromycin B [30].
2 Linearized plasmids should be dephosphorylated beforehand to ensure the isolation of
single-copy integrants.
PROTOCOL 7.1
Recombinase-Mediated Cassette Exchange
Equipment and reagents
•
Fluorescent flow cytometer (Becton Dickinson)
•
Dulbecco's modified Eagle's medium (DMEM; GIBCO)
•
Fetal bovine serum (FBS) (Wisent)
•
Gancyclovir (Sigma)
•
Hygromycin B (Roche)
•
Methocel (Fluka)
•
Salmon sperm DNA (Sigma)
•
Lipofectamine (Invitrogen).
Method
1 Mix slowly pL1-HYTK-L1 plasmids, in 1 : 1 or 1 : 2 ratio with Lipofectamine in 50
μ
ltotal
volume.
a
2 Incubate with cells for 4-6 h.
3 After 24-48 h post-lipofection, replace culture media by DMEM with 750
μ
g/ml of
hygromycin B.
