Cloning of the -cateninY654E-fl-neo targeting vector
A -catenin targeting vector was constructed using the TNLOX1-3 vector as basis. Pfu ultra
high fidelity DNA polymerase (Stratagene) was used to generate a 5.2 kb genomic fragment
containing exons 7-11, and a 3.3 kb genomic fragment containing exons 12-15. Using site
directed mutagenesis, the latter fragment was modified at amino acid residue 654 present
within exon 13, to replace the original tyrosine (Y) residue into a glutamic acid (E). Next,
both 5.2 and 3.3 kb wild type fragments, and the modified 3.3 kb fragment were cloned into
the TNLOX1-3 vector. To enable specific removal of the PGK-Neo cassette at a later stage,
we replaced the original Neo cassette with that of pL451 [1] in which the PGK-Neo cassette is
flanked by FRT sites. Further cloning details are available upon request. The final targeting
vector, Y654E-fl-neo depicted in Figure 1, was sequence-verified before gene targeting of
129Ola IB10 embryonic stem (ES) cells. Electroporation and G418 selection was carried out
as described before [2]. Southern blot in combination with PCR analysis of more than 2,000
selected ES clones, revealed that in 2 clones the -catenin locus was correctly targeted. One
of these clones showed a normal karyotype of 40 chromosomes and was used for injections.
Genotyping -cateninY654E mice
The chorion or tail tips were used for DNA isolation to determine the genotype of embryos or
neonates, respectively. The following primers were used: geno654-F ‘gattggagaccagaagccttg’
and geno654-R ‘acccaatgtaaagcatgacgtg’.
Intestinal tumour analysis
-cateninY654/Y654E-fl mice were mated with CAG-Cre mice to obtain -cateninY654/E654 mice
and wild-type littermates (mixed C57Bl/6J;129Ola background, F2-F3). These animals were
sacrificed at 16-18 months of age or when moribund. Apc+/15lox mice and Fabpl-Cre mice
1
were mated with -cateninY654/Y654E-fl-neo mice (mixed C57Bl/6J;129Ola background, F2-F3) to
obtain Apc+/15 mice, Apc+/15;-cateninY654/E654 mice, and control littermates. These animals
were sacrificed at 8-9 months of age or when moribund. All animals analyzed were wild-type
for the Cre construct. The entire intestines were removed, opened longitudinally and washed
with PBS. Sections of approximately 10cm were spread out flat on filter paper, fixed
overnight at 4oC in 10% phosphate-buffered formalin, and transferred to 70% ethanol. The
size and location of tumours was determined using a dissection microscope.
Fluorescent immunoblotting
Laemmli sample lysates were boiled for 5min prior to loading on polyacryl-amide gels.
Resolved proteins were transferred onto PVDF membranes (Immobilon-FL, Millipore), which
were than blocked in Odyssey-blocking buffer (Westburg) for 1hr at room temperature, and
incubated in primary antibody overnight at 4oC. Primary and secondary antibodies employed
for immunoblotting are depicted in the following table. Blots were scanned using an Odyssey
Imager (Li-Cor Biosciences) of which the results were analyzed using Odyssey Li-Cor
software.
Antibody
Purified mouse-anti--catenin
Polyclonal rabbit-anti-phospho--catenin (Ser675)
Polyclonal rabbit-anti-PAN-Cadherin
Monoclonal mouse-anti-N-Cadherin
Polyclonal rabbit-anti-phosphoCREB (S133)
Polyclonal rabbit-anti-GFP (A11122)
Polyclonal rabbit-anti-Tubulin
Mouse-anti-Tubulin
Dilution
1:2000
1:500
1:500
1:1000
1:250
1:500
1:10000
1:5000
Company
BD Transduction Laboratories
Cell Signaling
Cell Signaling
BD Transduction Laboratories
Cell Signaling
Molecular Probes
Abcam
Sigma Aldrich
Goat-anti-mouse IgG IRDye 680
Goat-anti-rabbit IgG IRDye 800CW
1:5000
1:5000
Westburg
Westburg
Primary and secondary antibodies immunohistochemistry
2
Antibody
Monoclonal mouse-anti-E-cadherin
Monoclonal rabbit-anti-β-catenin 1247-1
Polyclonal rabbit-anti-phosphoHistone H3 (Ser10)
Monoclonal rat-anti-CD44
Monoclonal rabbit-anti-Cyclin-D1
Dilution
1:1000
1:2000
1:1000
1:1000
1:200
Company
BD Transduction Laboratories
Epitomics
Millipore
BD Transduction Laboratories
Vector Laboratories
HRP-conjugated goat-anti-mouse IgG (EnVisionTM)
HRP-conjugated goat-anti-rabbit IgG (EnVisionTM)
HRP-conjugated goat-anti-rat
n/a
n/a
1:250
Dako
Dako
Jackson Immuno Research
References
1
Liu P, Jenkins NA, Copeland NG. A highly efficient recombineering-based method for
generating conditional knockout mutations. Genome Res 2003;13:476-84.
2
Smits R, Kielman MF, Breukel C, Zurcher C, Neufeld K, Jagmohan-Changur S, et al.
Apc1638T: a mouse model delineating critical domains of the adenomatous polyposis coli
protein involved in tumorigenesis and development. Genes Dev 1999;13:1309-21.
3