Melanoma transplants in "green" mice: Fluorescent cells in tumors are not equivalent to host-derived cells

Background: To examine the usefulness of green fluorescent protein (GFP) mice for studying the interactions between normal cells and tumor cells in a host, we used a melanoma model in such "green" mice [C57BL/6-Tg (CAG-EGFP)1Osb mice]. Mice were given a subcutaneous injection of B16-F10 cells, and the resultant primary tumors were removed. Then cells from individual tumors were cultured. Results: The proportion of EFGP+ cells was determined by fluorescence-activated cell sorting (FACS) and was 6.8% ± 3.2% (mean ± s.d.) on day 1 of culture, 0.6% ± 0.3% on day 2, and 0.02% ± 0.01% at day 7. In all cases, isolated cells grew at a constant rate, but fluorescence decreased over time and became undetectable on day 14. Cells were tested using PCR for the presence of an EGFP-specific sequence, and results were negative in all cases, thus indicating that the cells did not harbor the host's reporter gene. Cells were also tested for the presence of EGFP mRNA, which was consistently detected for 22 days after the start of culture. The tumorogenicity of the cultured cells was confirmed in GFP mice injected with cells from a selection of cultures. Conclusions: In a melanoma model in GFP mice, the detection of "green" cells in tumors was not equivalent to the detection of host-derived cells. Such "masking" was caused by a transient, but lasting, transfer of EGFP mRNA from the host's normal cells to tumor cells. Thus, an analysis of tumors postmortem by techniques that yield only a single snapshot can lead to incorrect interpretations and erroneous conclusions.

DNA from tissues of young mice is optimal for genotyping

Background Genotyping of mice is a common procedure in animal facilities. The aim of this study was to compare the quantity and quality of DNA extracted from samples obtained from young mice (YM; 10 d old) and adult mice (AM; 12 weeks old). We collected samples from the tail and ear of YM and AM. We also sampled blood, check cells (via buccal swabs), hair and fecal pellets of AM, and biopsied distal phalanx of YM. We isolated DNA using commercial kits and determined concentrations and purity by spectrophotometry. The integrity of DNA was evaluated by agarose-gel electrophoresis and polymerase chain reaction (PCR). Results DNA in all samples was amplified successfully but the intensities of bands after electrophoresis was heterogeneous. In general, tissues from YM yielded more DNA than those from AM, with differences being statistically significant for ear samples (38 ± 12 ng/μL for YM; 7 ± 3 ng/μL for AM; P = 0.006). In YM, the most DNA was obtained from ear and tail samples, with differences from the amounts obtained from phalanx samples being statistically significant (P = 0.02 and P = 0.005, respectively). In AM, the most DNA was obtained from tail and blood samples. Samples obtained by non-invasive sampling methods in adults resulted in a deficient DNA extraction. Conclusions The results of the present study do not support the previous recommendations for using non-invasive methods to genotype adult animals. The use of newborn tissue samples showed the highest efficiency for DNA extraction.