Whole endoderm explants were isolated from stage 10.5 by removing the surrounding ecto-and mesodermal tissue using forceps and transferring the endodermal cone to an 0.7
% agarose dish. Dorsal (DE) and ventral (VE) endoderm were distinguished according to the position of the blastopore lip. Explants were cultured in 1× MBStill control siblings
2.2.1.6 Chemical treatments of embryos and explants
Retinoic acid (RA)Whole embryos stage 8-9, endodermal explants or animal caps were incubated in 5 µM5 µM RA (all-trans-RA) in corresponding buffer for 1 hr. After treatment, embryos and explants1 hr. After treatment, embryos and explants were cultured in their buffer till control siblings reached the desired developmental stage.
Control treatment was done with corresponding amount of EtOH (Chen et al., 2004).
RA: 10 mM stock in 100% EtOH, store at -20°C, light sensitive; SigmaSigma
BMS453
Whole embryos stage 8-9, endodermal explants or animal caps were incubated in 1µM BMS4531 µM BMS453 for 1 hr. After treatment, embryos and explants were cultured in 0.8× MBS till control sib-0.8× MBS till control sib-MBS till control sib-till control sib-lings reached the desired developmental stage. Control treatment was done corresponding amount of DMSO (Chen et al., 2004).
BMS 453: 10 mM stock in DMSO, store at -20°C, light sensitive;light sensitive;
gift from Bristol Myers Squibb
Dexamethason (Dex)
Proteins fused to the glucocorticoid receptor (GR) binding domain are immobilised in the(GR) binding domain are immobilised in thebinding domain are immobilised in the cytoplasm by heat shock poteins. Dexamethason, a steroid hormone, compatitively binds to the GR domain releasing the protein from its heat shock inhibitors.
Dex (1000×):1000×):: 8 mg/ml in 100% EtOH, store at -20°C, dilute 1:1000;
2.2.2 Bacterial work
2.2.2.1 Generation of chemical competent cells
To obtain chemical competent cells (Hanahan et al., 1991), a 5 ml oN culture was(Hanahan et al., 1991), a 5 ml oN culture was, a 5 ml oN culture was inocula-ted and diluinocula-ted into 300 ml prewarmed LB-medium in a 2 l Erlenmeyer flask, shaking at 220 rpm. When the culture reached OD600= 0.3, cells were grown at 18°C until ODat 18°C until OD600= 0.4−
0.5 (exponential growth phase). The culture was split in 50 ml falcon tubes and centrifugedexponential growth phase). The culture was split in 50 ml falcon tubes and centrifuged at 4°C and 6000 × g for 10 min. Working on ice, the supernatant was decanted, the pellet resuspended in 15 ml prechilled transformation buffer and incubated on ice for 15 min The suspension of three tubes was pooled and re-centrifuged for 5 min at 4°C, 6000 × g.
The pellet was resolved in 15 ml transformation buffer and supplied with 525 µl DMSO, incubated on ice for 5 min and supplied again with 525 µl DMSO. After an incubation of 5 min 200 µl aliquots were shock-frozen in liquid nitrogen and stored at -80°C .200 µl aliquots were shock-frozen in liquid nitrogen and stored at -80°C .aliquots were shock-frozen in liquid nitrogen and stored at -80°C .
LB- Medium: 1% (w/v) Bacto-Trypton (Difco), 0.5% (w/v) �east/v) Bacto-Trypton (Difco), 0.5% (w/v) �eastv) Bacto-Trypton (Difco), 0.5% (w/v) �east/v) �eastv) �east extract (Dif-co), 1% (w/v) NaCl, pH 7.5./v) NaCl, pH 7.5.v) NaCl, pH 7.5.
Transformation buffer: 10 mM PIPES (pH 6.7 with KOH), 15 mM CaCl2, 250 mM KCl, addition of +55 mM MnCl2 prior to usage
2.2.2.2 Chemical transformation of competent cells
An 200 µl cell aliquot was unfrozen on ice. 5-10 ng of plasmid- DNA or 1 µl ligation mix-ture were added and cells incubated for 30 min on ice. DNA uptake was induced upon heat shock at 42°C for 50 s followed by immediate incubation on ice for 3for 50 s followed by immediate incubation on ice for 3followed by immediate incubation on ice for 3 min. Bacteria were directly spread onto prewarmed LB-agarose plates using small glass beads and incubated oN at 37°C in an incubator. LB-plates contained antibiotics for selectionantibiotics for selection (see 2.1.6) and, in, in need of blue-white screening, they were preincubated at 37°C with 40 µl X-Gal and 100µlat 37°C with 40 µl X-Gal and 100µlwith 40 µl X-Gal and 100µl IPTG.
LB- Agar: 1.5% (w/v) Agar (Difco) in LB-media
X-Gal: 20 mg/ml X-Gal in Dimethylformamid, store –20°C; Sigma IPTG: 100 mM IPTG in dH2O, store –20°C; Roth
2.2.2.3 Glycerol stocks
A glycerol stock was made for long time storage of bacterial culture. 1 ml of oN cultureof bacterial culture. 1 ml of oN culture. 1 ml of oN culture was spin down for 2 min in a table centrifuge. The pellet was resuspended in 500 µl fresh LB-media. 250 µl of 100% glycerol was added and the solution mixed on a rotor for 10 min.
The glycerol stock was stored at -80°C.
2.2.3 DNA work (according to Sambrook et al., 1989) 2.2.3.1 Nati�e agarose gel electrophoresis
1%− 2% (w/v) agarose gels were prepared in 1× TAE and supplied with 0.5 µg/ml EtBr.
Analytic samples were mixed on parafilm with a drop of DNA loading dye prior to laodingon parafilm with a drop of DNA loading dye prior to laodingwith a drop of DNA loading dye prior to laoding the sample to the gel pocket. Gels were run in 1× TAE (8in 1× TAE (8(8 min, 100 mA) in a home made horizontal electrophorese chamber. The negative charged DNA fragments migrate in an electric field from anode to the kathode thereby separating according to their size.Frag-ment sizes were estimated in comparison to a molecular standard marker (Low-, Middle-, High range ladder, MBI- Fermentas). Images were taken with a video documentation sy-stem (Chemidoc, Biorad).
Glycerol loading buffer: 10 mM Tris-HCl pH 7.5, 10 mM EDTA pH 8, 0.025%
(w/v) Bromphenol blue, 0.025% (w/v) Xylencyanol, 30%
(v/v) Glycerol 99%
50× TAE buffer: 2 M TrisHCl, 1M acetat, 0.1 M EDTA 0.5 M EDTA 0.5 M Na2EDTA*2H2O, pH 8
2.2.3.2 DNA purification Mini plasmid purification
Small amounts of plasmid were isolated using the IllustraTM plasmid spin Mini Kit accor-ding to the manufacturers protocol. 5 ml bacterial culture was spin down, resuspended and lysed in an alkaline buffer including RNase A. After 5 min incubation at RT the reaction was stoped by addition of a neutralisation buffer. Precipitate including lipids and denatured proteins, was spin down for 10 min. The supernatant was applied to the column and spin down whereby DNA bound to the column matrix. Salts were washed away using a EtOH containing buffer. Finally the DNA was eluted from the column in 50 µl elution buffer (1M(1M TrisHCL, pH 8) or dH2O. Plasmids were confirmed by sequencing and stored at -20°C.. Plasmids were confirmed by sequencing and stored at -20°C.