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A very simple procedure for the isolation of high-quality, high-molecular-weight genomic DNA from embryonic stem cells is described. The DNA is very stable once dried and can be stored for long periods of time without refrigeration. Living cells are lysed in a sodium dodecyl sulfate and EDTA buffer containing proteinase K and then air-dried. Samples can be processed in bulk, and an individual can easily process thousands of samples for extraction and shipment on a daily basis using only common laboratory materials such as plastic ware and a multichannel pipetteman.
Work with embryonic stem (ES) cells has been increasing at a significant rate over the last several years and will likely continue to do so for the foreseeable future. One of the principal uses of murine ES cells is the generation of genetically altered cell lines that can be subsequently used to study the genome alteration in mice following germline transmission. To make genome-altered mice by homologous recombination gene targeting technology requires electroporation of ES cells with the targeting vector, then picking anywhere from 200 to 5000 selection-resistant (G418, Hygromycin) clones, expansion, and analysis of DNA for identifying the clones containing the targeted allele.1,2 Because of the sometimes very large number of ES clones picked, the completion of the DNA analysis requires several weeks and storage of samples for later DNA analysis. We have developed an easy storage procedure to handle a large number of cell samples that overcomes the need to extract DNA immediately after cell lysis or to store samples at 4°C or −20°C thus freeing up valuable freezer space. Moreover, the procedure allows storage of lysed ES cell samples directly in the same culture dishes where the cells were grown. The ES cell DNA samples are resistant to exposure to elevated temperatures (e.g., 37°C) for extended periods of time. These features make easier both the management of scheduled DNA extractions and the shipment of ES cell DNA samples via standard post or courier service for analysis at a distant site of a collaborator.
In a test experiment we compared the quality of genomic DNA extracted from ES cells that were stored for varying lengths of time at two different temperatures: 22°C (room temperature) and 37°C. In brief, 10 cultures of ES cells were grown to confluence in 35-mm dishes (the procedure works equally well using 6-, 24-, or 96-well plates). Each culture contains the same heterozygous ES clone, named FIG25, which we generated in a knockout experiment by insertion of the IRES/hrGFP (humanized Renilla green fluorescent protein) reporter cassette (Stratagene) and the neomycin-resistant PGK-neo cassette3 flanked by two loxP sites in a single-copy gene.4 The IRES sequence allows for cap-independent translation initiation of reporter cassettes.5 After removing the media, 0.5 mL of lysis buffer (50 mM Tris-HCl [pH 8.0], 5 mM EDTA [pH 8.0], 200 mM NaCl, 1% [w/v] SDS) containing proteinase K to a final concentration of 0.1 mg/mL, was added directly to the ES cells. The cells were digested in the original culture dishes overnight in either a 37°C air oven or a 37°C CO2-humidified cell culture incubator. Afterwards, lysed ES cell samples were divided into two groups that were incubated at two different temperatures. The dishes containing samples of group 1 were incubated at 22°C (room temperature) for the following lengths of time: sample 1 for 1 d; sample 2 for 3 d; sample 3 for 10 d; sample 4 for 30 d; sample 5 for 90 d. The dishes containing the samples of group 2 were incubated at 37°C for the following lengths of time: sample 6 for 1 d; sample 7 for 3 d; sample 8 for 10 d; sample 9 for 30 d; sample 10 for 90 d. At both temperatures, the samples dried out after 24 h.
The stability of DNA in these dried samples was tested in a long-distance shipment. Samples were sent out in the original culture dishes placed in a standard cardboard mailing envelope. They reached the receiving laboratory after 10 d in transit. Genomic DNA was purified by adding 500 μL of TE (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]), followed by a single extraction with an equal volume of standard phenol:chloroform (1:1), followed by ethanol precipitation with 1 mL of 95% EtOH. The precipitated DNA was either immediately fished out with a 20-μL pipette tip or pelleted and then dissolved in 0.1 mL of TE. Southern blot hybridization and PCR reactions were used to test the quality of the DNA preparations. For Southern blot hybridization, 10 μL of each DNA sample, corresponding to about 10 μg, were digested with EcoRI and run on an agarose gel (Figure 1A1A).). DNA was transferred from the gel to Hybond-N+ nylon membrane by standard capillary alkaline blotting and then fixed to the membrane by baking for 2 h at 80°C. Figure 1B1B shows the results of the hybridization of the membrane with a 32P-labeled probe 1.5 kb in length, containing the IRES/ hrGFP cassette. After hybridization, the membrane was exposed to X-ray film for 5 d at −70°C with an intensifying screen. The 1.5-kb hybridizing band, which marks the mutated form of the allele, was detected in all the DNAs isolated from ES cell samples (Figure 1B1B).
In conclusion, both photography of the ethidium bromide–stained DNA gel (Figure 1A1A)) and the result of Southern blot hybridization (Figure 1B1B)) are evidence that high-yield and high-molecular-weight genomic DNA were obtained with our procedure even when DNA samples were stored dried for 90 d at either 22°C or 37°C. Figure 1C1C shows the result of a PCR analysis. Using standard procedures, PCR reactions were carried out with 1 μL of ES cell genomic DNA (samples 1 to 10) as template in a total reaction volume of 50 μL. The following oligonucleotide primers were used to amplify a 200-bp fragment from the mutant allele containing the region between hrGFP and the PGK-neo cassette: forward (hrGFP primer) reverse (PGK-neo primer) -CAGACTGCCTTG 3′. As a out a PCR reaction with no genomic DNA template. PCR conditions were 40 cycles, annealing temperature 55°C. The PCR products were analyzed by agarose gel electrophoresis and EtBr staining (Figure 1C1C).). The result was that all the DNA samples from ES cells yielded the predicted 200-bp amplified fragment. Thus, we concluded that DNAs isolated and stored according to our procedure could be used for PCR.
Lastly, we successfully used this procedure in a gene-knockout experiment to transfer to Europe, via the United States postal system, 200 samples of ES cell clones for DNA screening. In this case, the DNAs were stored dried for 10 d at room temperature in 96-well culture dishes, and the DNA was analyzed by Southern blot hybridization (data not shown). In conclusion, the procedure here describes a simple and rapid method for extracting, storing, and shipping high-quality ES cell DNA.
The financial support of Telethon-Italy (grant GP0283Y01) to C.T. and NIH grants AR46471 and DE13741 to T.L. are gratefully acknowledged. The authors declare they have no significant competing financial, professional or personal interests that might have influenced the performance or presentation of the work described in this manuscript.