Cell Line Mouse Strain Reference Transgenic method Culture method

Oct4 Inducible ES[MC1R(20):tetNr0b1(15)] Munoz Descalzo et al., Tet-repressible Nishiyama et al., 2009
Null AG23191 Mus musculus 2013 gene expression DMEM containing 15% FBS, 1000 U/ml LIF
129S6/SvEvTac. Faunes et al., 2013 system (Masui et (Millipore), 1 mM sodium pyruvate, 0.1 mM MEM
Nishiyama et al., 2009 al., 2005). non-essential amino acids, 2 mM glutamate,
0.1 mM ß-mercaptoethanol.

The gene induction and other experimental work

were carried out using a gelatin-coated dish without
feeder cells.

Mouse embryonic fibroblast (MEF) cells were

derived from 12.5 dpc hybrid embryo of
Tg(DR4)1Jae/J and ICR (Tucker et al., 1997) and
were cultured in DMEM containing 10% FBS, 1 mM
sodium pyruvate, 0.1 mM MEM non-essential
amino acids, 2 mM glutamate, 0.1 mM ß-
mercaptoethanol. MEF cells were treated with
10µg/ml Mitomycin C (Sigma) for 2 hours and used
as feeder cells or stored.

ES cells were cultured on a feeder cells in medium

containing 0.2µg/ml Dox and 1.0μg/ml puromycin
for 6 days and further cultured on gelatin coated
dish in medium containing 0.2μg/ml Dox and
1.5μg/ml puromycin for 3 days. One day before
transgene induction, ES cells were plated onto a
gelatin coated 6 well plate at 2x105 cells/well for 24
hours of transgene induction, 1x105 cells/well for 48
hours, and 0.5x105 cells/well for 72 hours. Dox was
removed by washing the cells three times with PBS
at interval of 3 hours.
Tet-OFF system (For AG23191 cell line)

In the absence of Tc or its analog Dox, transgene is turned on.

Tc-regulatable transactivator (tTA) binds to the hCMV*-1 promoter. The promoter consists of a Tc-responsive element (TRE) upstream of a minimal
promoter of the human cytomegalovirus (hCMV) immediate early gene. tTA is a fusion protein consisting of a TRE binding domain and the herpes simplex
virus VP16 activation domain.

(C.f. Tet-ON: rtTA binds TRE, and induces transcription in the presence of Dox. See Masui et al., 2005 for difficulties with this system, and the advantages of
Tet-off over Tet-on)

ROSA-26 locus:

Identified as a gene-trapped locus on chromosome 6, where B-geo was shown to be expressed ubiquitously throughout the body of the mouse.

Usefulness: this locus is one where proteins can be expressed ubiquitously at a moderate level.

Recombinase-mediated cassette exchange (RMCE) system:

Alternative to homoologous recombination for the efficient introduction of a transgene into a cassette acceptor (CA) locus. In brief, the LoxP and LoxPV
sequences on a transfected each recombines specifically with the same lox sequence on the CA locus, upon transfection with a Cre expression vector.