2. Precancerous epithelial cells isolated from mouse mammary gland grow best in
ID: 271301 • Letter: 2
Question
2. Precancerous epithelial cells isolated from mouse mammary gland grow best in culture media supplemented with high levels of bovine serum (note: serum is plasma minus clotting factors). Culture in a defined medium, containing no serum, results in cessation of growth. However supplementation of this minimal medium with medium in which mouse mammary fat pads have been cultured (i.e., adipocyte-conditioned medium) results in initiation of DNA synthesis in the mammary epithelial cells. (In the organism, the mammary fat pad acts as a source of lipid, which is used by the mammary epithelial cells as an energy source as well as a source of lipid for milk production.) The factor in the conditioned medium that is responsible for the stimulation of DNA synthesis is stable after heating to 100 °C and after treatment with trypsin at 37 °C for 2 h. Answer the following questions: a) Based on this information, propose a hypothesis regarding the identity of the stimulatory factor, and b) What experiments might you perform to prove or disprove your hypothesis?Explanation / Answer
a)The hypothesis could be: If the stimulatory factor is a thermophilic DNA polymerase enzyme, then it is stable after heating at 100oC and after treatment with trypsin at 37oC for 2 h and stimulates DNA synthesis also.
b)To prove my hypothesis that the stimulatory factor initiates DNA synthesis, I would culture cells with stimulatory factor and without stimulatory factor.Then, I would isolate DNA from both the cultured cells separately.Then I would quantify the DNA isolated.The one with stimulatory factor would show increased DNA quantity in comparision to one without it.
To prove its thermophilic nature, I would take the adipocyte conditioned medium with the stimulatory factor in it and heat the medium to 100oC and treat it with trypsin at 37oC for 2 hours.Then,I would use two cell culture sets:One culture cells set would be left to grow in medium with stimulatory factor with no heat treatment and trypsin treatment and other culture cell set with medium with stimulatory factor exposed to heat treatment and trypsin treatment.Then isolate DNA from both culture sets separately and quantify the DNA.The DNA quantified would be almost the same as treatment does not affect the property of the stimulatory factor.
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