Preparation of the 1lox/2lox standard
1lox and 2lox specific PCR products (1lox-forward primer: GCCGGCTTTTCCTCA GACAGTGGAGATAGC, 1lox-reverse primer: CCTGTGTGCAGCAGACACTTCT TTGGCGTC; 2lox-forward primer: CCTCTGGGGATTAAACTCTTGGCCAGCCC, 2lox-reverse primer = 1lox-reverse primer) were generated using genomic DNA from Dnmt3a(2lox/2lox), Fabpl4× at -132-Cre(+/-), APC(Min/+) transgenic mice. PCR products were then purified using agarose gel-electrophoresis followed by gel extraction (QIAGEN Minielute Gelextraction Kit) and cloned into TA vectors (TOPO-cloning Kit, Invitrogen). Minipreps were prepared from individual colonies resulting from both 1lox and 2lox ligations and verified by sequencing. Plasmids with verified sequences were digested with EcoRI to release fragments containing the 1lox and 2lox specific inserts, respectively. Following gel electrophoresis and gel extraction the concentration of resulting 1lox and 2lox fragments were measured using capillary electrophoresis (Agilent, High sensitivity DNA Assay) and 1lox-2lox mixtures were prepared at variable molar ratios (1/9, 2/8, 3/7, 4/6, 5/5, 6/4, 7/3, 2/8, 1/9). The 1lox/2lox ratios of these final mixtures were verified again using capillary electrophoresis.
Probe based Real-Time PCR
The hydrolysis probe based Real-Time PCR reaction was performed as follows: 1lox- and 2lox reactions were conducted separately in 10 μl reactions containing 0.25 μM each of forward and reverse primers (1lox-forward primer: TAATCCCAGCACTGC ACTCA, 1lox-reverse primer: TTCTTTGGCGTCAATCATCA; 2lox-forward primer: CCTCTGGGGATTAAACTCTTGGCCAGCCC, 2lox-reverse primer: CCTGTGTGC AGCAGACACTTCTTTGGCGTC), 0.05 μl Universal Probe #69 (Roche Applied Science), LightCycler 480 Probes Master Mix (Roche Applied Science) and 50ng genomic DNA or 4.3pg of the 1lox/2lox standard. The cycling conditions were as follows: Preincubation at 95°C for 10min, then 45 cycles of 95°C for 10sec, 58°C for 45sec and 72°C for 1sec. Each reaction was measured in triplicates. For data analysis we used "LightCycler(r) 480 Software release 1.5.0 SP3" (Roche Applied Science) and the "Advanced Relative Quantification" module where the crossing points (Cp = Ct) were determined using the "2nd Derivative Maximum Method". The mentioned module quantifies 1lox signal (T, Target) relative to 2lox signal (R, Reference), considering the respective amplification efficiencies (E) using the following formula: E
CpR. The efficiencies were determined using the slope of the respective standard curve (E = 10-1/slope) with Cp values plotted against the logarithm of different dilutions. As dilution standards we used the incremental 1lox/2lox standard mixtures (10 standards, 10% dilution steps), in other words the samples themselves were used for calculation of reaction efficiencies. As a final control the PCR product was analyzed using agarose gel electrophoresis.
SYBR Green based Real-Time PCR
Real-Time PCR was performed using equipment of Roche Applied Science (LightCycler 480 SYBR Green I Master, Light Cycler 480). 1lox and 2lox-reactions were conducted separately in 10 μl volume containing 50ng DNA and 0.25 μM each of forward and reverse primers (1lox-forward primer: TAATCCCAGCACTGCACTCA, 1lox-reverse primer: TTCTTTGGCGTCAATCATCA; 2lox-forward primer: CCGAT GCAGACAGCCTCAGC, 2lox-reverse primer: CTTGTCACTAACGCCCATGG CCA). The following cycling conditions were chosen: Preincubation at 95°C for 10min, then 45 cycles of 95°C for 10sec, 60°C for 15sec and 72°C for 30sec. Each reaction was measured in triplicates. Data analysis was executed in analogy to Probe based Real-Time PCR. As a final control the PCR product was analyzed using agarose gel electrophoresis.
The Dnmt3a(2lox/2lox), Fabpl4× at -132-Cre(+/-), APC(Min/+) transgenic mice were generated as previously described for Dnmt3b(2lox/2lox), Fabpl4× at -132-Cre(+/-), APC(Min/+) mice [5, 6]. Briefly the conditional Dnmt3a allele contains loxP sites located in intron 17 and intron 20 of the endogenous Dnmt3a allele. Cre mediated excision results in deletion of exons 18-20, resulting in deletion of the catalytic domain. For a detailed description of the Dnmt3a (2lox/2lox) mice see , for details on Fabpl4× at -132-Cre(+/-) mice see .
No animal experiments were conducted with the intention of generating material for the present study. All mouse tissues and DNA analyzed in the present study were harvested for the purpose of an alternative, ongoing project analyzing the role of DNA Methyltransferase 3a in mouse tumorigenesis. All animal experiments of that project were approved by the MIT Department of Comparative Medicine, Boston, USA and were executed according to the institutional guidelines.
Harvesting of epithelial cells of the small intestine
Intestinal epithelial cells were harvested as described previously . Briefly, the small intestine was dissected from mice, divided into three thirds of equal length (proximal, middle, distal), rinsed and incubated in 20 ml harvesting buffer (3 mmol/L ethylenediaminetetraacetic acid (EDTA) plus 0.5 mmol/L dithiothreitol in PBS) at 37°C, rotating at 250 rpm for 30 min. Following incubation the intestinal tube was removed and floating epithelial cells centrifuged at 200×g and 4°C for 4 min. After removal of the supernatant, cell pellets were frozen at -80°C.
DNA of the harvested small intestine epithelial cells was isolated using the QIAGEN DNeasy Kit using the manufacturer's protocol.