2 Experimental Procedures
2.1. Mice
All experiments were carried out in accordance with the home office regulations.
The AhCre transgenic mouse, where expression of Cre recombinase can be
induced in gastrointestinal tract by -naphthoflavone, was generated by Heather
Ireland (Ireland et al., 2004) and AhCreER(T) mouse, which has the additional
control over Cre activity via Tamoxifen binding, was created by (Kemp et al.,
2004). BRG1 floxed mouse with loxP sites flanking exons 2 and 3 of BRG1 was
provided by Pierre Chambon (Sumi-Ichinose et al., 1997). APC mouse with loxP
sites located in the introns around APC exon 14 was generated by (Shibata et al.,
1997). Villin-Cre-ERT2 mouse expressing a tamoxifen-dependent Cre recombinase
under control of the villin promoter was constructed by (el Marjou et al., 2004).
And finally BLG1-Cre mouse bearing Cre recombinase under the regulation of the
mammary gland specific beta-lactoglobulin promoter was created by (Selbert et
al., 1998).
All mice were fed the Harlan standard diet (scientific died services) and water was
provided ad libitum.
2.2 Genotyping
Mice were genotyped by PCR using DNA extracted from tail biopsies at weaning
age (four weeks) and confirmed at death. All primers used were designed by
‘Primer3’ software at http://fokker.wi.mit.edu/primer3/input.htm, checked for
specificity using Ensembl and ordered from ‘Sigma Genosys’. PCR was carried
out in 96-well plates containing 47.5 l master mix (all components were
purchased from Promega unless stated otherwise) and 2.5 l DNA sample using
Techne Flexigene thermocycler. 25 l of PCR products were run on a 2% agarose
gel (4 g agarose, 200 ml 1X TBE buffer (Sigma), 10 l ethidium bromide) at
120 V.
2.2.1 DNA purification (Puregene method)
A small section of the tail (3-5 mm long) was obtained from either dead or
properly anaesthetised mouse and stored at -20C in 1.5 ml eppendorf tube. For
DNA isolation 500 l of Cell Lysis Solution and 10 l of 20 mg/ml Proteinase K
(Roche) were added to the tissue and incubated overnight at 37C under agitation.
The next day the content of the tubes was cooled to the room temperature, mixed
with 200 l of Protein Precipitation Solution (Gentra) and centrifuged at 13,000
rpm for 10 min. The supernatant was mixed with 500 l of isopropanol in a fresh
1.5 eppendorf tube and spun at 13,000 rpm for 15 min. The supernatant was
carefully discarded and the tubes were left to dry on air for 1 hour. DNA was then
dissolved in 500 l of nuclease-free water and 2.5 l of the resulting solution was
used in PCR reactions.
2.2.2 Cre/LacZ combined PCR
The master mix contained 31.7 l H2O, 10 l 5X Colourless GoTaq Flexi buffer,
5 l 25 mM MgCl2, 0.4 l 25 mM dNTPs (Bioline), 0.2 l GoTaq DNA
Polymerase (Promega), 0.1 l 100 M forward primer and 0.1 l 100 M reverse
primer. The PCR conditions were 95C for 3 min, 95C for 30 sec, 55C for 30
sec, 72C for 1 min, repeat step 2-4 for 30 cycles, 72C for 5 min and 15C hold.
The Cre forward primer 5’-TGACCGTACACCAAAATTTG-3’ and the reverse
primer 3’-ATTGCCCCTGTTTCACTATC-5' yield the PCR product of 1000 bp,
whereas the LacZ forward primer 5’- CTGGCGTTACCCAACTTAAT-3’ and
reverse primer 3’-ATAACTGCCGTCACTCCAAC-5’ produce a fragment of
500 bp.
2.2.3 APC PCR
The master mix contained 31.7 l H2O, 10 l 5X Green GoTaq Flexi buffer, 5 l
25 mM MgCl2, 0.4 l 25 mM dNTPs (Bioline), 0.2 l DreamTaq DNA
Polymerase (Fermentas), 0.1 l 100 M forward primer and 0.1 l 100 M reverse
primer. The PCR conditions were 95C for 3 min, 95C for 30 sec, 60C for 30
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sec, 72C for 1 min, repeat step 2-4 for 29 cycles, 72C for 5 min a 15C hold. The
forward primer 5’-GTTCTGTATCATGGAAAGATAGGTGGTC-'3 and the
reverse primer 3’-CACTCAAAACGCTTTTGAGGGTTGATTC-'5 generate 226
bp and 314 bp products for the wild type and floxed allele respectively.
2.2.4 AhCre non responder PCR
The master mix contained 31.7 l H2O, 10 l 5X Green GoTaq Flexi buffer, 5 l
25 mM MgCl2, 0.4 l 25 mM dNTPs (Bioline), 0.2 l DreamTaq DNA
Polymerase (Fermentas), 0.1 l 100 M forward primer and 0.1 l 100 M reverse
primer. The PCR conditions were 94C for 5 min, 94C for 20 sec, 57C for 20
sec, 72C for 30 sec, repeat step 2-4 for 35 cycles, 72C for 5 min a 15C hold.
The forward primer 5’-AGGTTCCCTGGGACTTGTTT-'3 and the reverse primer
3’-TCACCAAACCCTCCATCAGT-'5 produce 196 bp and 178 bp products for
the responder and non responder allele respectively.
2.2.5 BRG1 PCR
The master mix contained 31.7 l H2O, 10 l 5X Green GoTaq Flexi buffer, 5 l
25 mM MgCl2, 0.4 l 25 mM dNTPs (Bioline), 0.2 l DreamTaq DNA
Polymerase (Fermentas), 0.1 l 100 M forward primer and 0.1 l 100 M reverse
primer. The PCR conditions were 94C for 2.30 min, 94C for 30 sec, 60C for 30
sec, 72C for 1 min, repeat step 2-4 for 35 cycles, 72C for 5 min a 15C hold. The
forward primer 5’-CCAAGGTAGCGTGTCCTCAT-'3 and the reverse primer
5’-CACTGCTCAGCTTCACTTGC-'3 produce 407 bp and 500 bp products for the
wild type and floxed allele respectively.
2.3 Tissue harvesting
The gut was dissected using a micro-dissection kit. Fur was sprayed with ethanol
and an incision made along the midline of the abdomen. Gut was flushed with
water and samples of small intestine (four or three 1.5 cm pieces) were taken at
approximately 7 and 20 cm away from pyloric junction. The small intestine
sections were wrapped together longitudinally in surgical tape as well as sections
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of colon. Samples from liver, pancreas, spleen, pyloric junction, kidney and
bladder were also harvested. Once collected, the samples were fixed in ice cold 4%
formalin fixative and incubated for 24 hours prior to transfer into ice cold 70%
ethanol for short-term storage before embedding.
2.4 Processing tissue for light microscopy
Once fixed, tissues were transferred to a histology unit, where they were embedded
in paraffin wax, sectioned at 5 m and placed onto a glass microscope slide (plain
slides for H&E staining or poly-L-lysine coated slides for immunohistochemistry).
2.5 Immunohistochemistry
For all IHC procedures the tissue sections were de-waxed and rehydrated using
following protocol: two 5 min washes in Xylene, two 3 min washes in 100%
ethanol, one 3 min wash in 95% ethanol and one 3 min wash in 70% ethanol prior
to transfer into dH2O. Area containing tissue was drawn around with DAKO water
resistant pen. Incubation steps were carried out in humidifying box at room
temperature (unless stated otherwise). Serum (DAKO, goat #X0907 and rabbit
#X0902) dilutions were prepared in TBS/Tween 1% and the same buffer was used
for three 5 min washes between incubations. Before mounting, sections were
shortly stained with Alcian Blue by 10 sec incubation in Alcian Blue staining
solution (1 g Alcian Blue in 100 ml of 3% Acetic Acid, pH 2.5) to reveal Goblet
cells, washed in running tap water, briefly counterstained in Haematoxylin
(Mayer’s Haemalum, blue cytoplasmic stain) for 30 sec and washed under running
water for 5 min. Washed slides were dehydrated by following rehydration protocol
in reverse and mounted using DPX. Sections were not allowed to dry at any point
during the procedure.
2.5.1 BRG1 (SWI/SNF remodeling complex ATPase subunit)
Sections were boiled in 10mM citric buffer (pH 6) for 20 min, cooled for 30 min
and incubated with peroxidase block (Mouse Envision Kit, DAKO #4006) for 5
min. Sections were washed in TBS/T, incubated in 10% normal rabbit serum for 30
min followed by 1 hour incubation with the mouse monoclonal primary BRG1 G-7
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antibody (1:200, Santa Cruz Biotechnology, sc-17796) in 10% normal rabbit serum
(alternatively incubation with primary antibody could be carried out overnight at
4C). Sections were washed in TBS/T and incubated with labelled polymer-HRP
anti-mouse antibody (Mouse Envision Kit, DAKO #4006) for 1 hour. Sections
were washed in TBS/T and stained with DAB solution (1 drop of DAB chromogen
per 1 ml of DAB substrate buffer, Mouse Envision Kit, DAKO #4006) for 5-10
min until the sufficient staining level.
2.5.2 -catenin (Wnt signalling component)
Sections were boiled in 10mM citric buffer (pH 6) for 20 min, cooled for 30 min
and incubated with peroxidase block (Mouse Envision Kit, DAKO #4006) for 5
min. Sections were washed in TBS/T, incubated in 10% normal rabbit serum for 30
min followed by 1 hour incubation with the mouse monoclonal primary -catenin
antibody (1:100, Transduction Laboratories, #610154) in 10% normal rabbit serum
(alternatively incubation with primary antibody could be carried out overnight at
4C). Sections were washed in TBS/T and incubated with labelled polymer-HRP
anti-mouse antibody (Mouse Envision Kit, DAKO #4006) for 1 hour. Sections
were washed in TBS/T and stained with DAB solution (1 drop of DAB chromogen
per 1 ml of DAB substrate buffer, Mouse Envision Kit, DAKO #4006) for 5-10
min until the sufficient staining level.
2.5.3 CD44 (cell adhesion, Wnt pathway target gene)
Slides were boiled in 10mM citric buffer (pH 6) for 20 min, cooled for 30 min and
blocked for 20 min in 1.5% H2O2 (Sigma) in TBS/T. Sections were washed in
TBS/T, blocked in 10% normal rabbit serum for 20 min and followed by 1 hour
incubation with rat anti-mouse CD44 primary antibody (1:50, Pharmigen,
#550538). Slides were washed, incubated with secondary biotinylated rabbit antirat antibody (1:200, DAKO, #E0468) for 30 min, washed and incubated with
ABC-HRP (Vectastatin Elite ABC standard kit #PK6100) for 30 min. Finally,
sections were washed and stained with DAB (DAKO #K3467).
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2.6 LacZ staining of the small intestine
Small intestine was removed and flushed with ice cold 1X PBS and X-Gal Fix (2%
formaldehyde, 0.1% glutaraldehyde in PBS). 7 cm sections of the small intestine were placed on
the wax plate with the mesenteric line uppermost, cut open, spread and pinned down. Tissue was
then fixed in X-Gal Fix for at least 1 hour, washed in 1X PBS and incubated in demucifying
solution (5 ml glycerol, 5 ml 0.1M Tris (pH 8.2), 10 ml ethanol, 30 ml 0.9% NaCl, 170 mg DTT)
for 1 hour. Mucin was blown off the tissue using plastic Pasteur pipette in 1X PBS and sections
were incubated with X-Gal Staining solution (0.1 g MgCl, 0.48 g potassium ferricyanide, 0.64 g
potassium ferrocyanide and 2 ml X-Gal (Promega, #V3941) in 500mls PBS) until the sufficient
staining was achieved. Once stained, tissues were washed with 1X PBS and fixed with formalin
to avoid further staining.
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