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BIOL 598 Advanced Topics in Gene Expression
Annotation of Figure
This figure depicts a northern blot showing TERT mRNA expression in a variety of tissues from mTERT Tg mice and the wild type mice. Twenty micrograms of tissue were collected from the thymus, lung, liver, kidney, mammary gland (10 micrograms were collected from the mammary gland) as seen in figure A and twenty micrograms from the heart, muscle, brain, testis, and MEF's which are mice embryonic fibroblasts. The northern blot clearly shows increased mTERT mRNA expression in all organs and MEF's as compared to the corresponding wild type mice. This ensures that the experimental mTERT mice shows a higher level of mTERM mRNA levels. The next figure serves to display if a correlation is associated between mTERT RNA levels and telomerase activity.
Panel A displays a line graph showing that there is an association between TERT transgene expression and breast cancer in aging mice. This graph depicts mTERT mice in black diamonds and non-Tg littermate control mice with grey triangles. Using a variety of phenotypic markers and histopathology to detect cancer, researchers were able to monitor the development of cancers over a time frame of 125 weeks.
In this part of the figure, pictures were taken to show how histopathology was used to detect the presence of cancer in the tissues. These cancers were invasive, depicted histological patterns differently, and displayed malignant characteristics such as nuclear pleomorphism, anisocytosis, increased nuclear to cytoplasmic ratios, and increased levels of mitotic activity. The different panels outline markers such as necrosis (unnatural death of cells) (B), squamous differentiation (C), cribiforms (D), acinar- berry shaped formation of an exocrine gland (E/H), solid invasive breast cancer (F/I), preinvasive MIN lesions (G/J), mitotic figures (H/J), and in situ lesions (G).
Figure 3A 3B
The above figure displays telomere lengths and proliferation assays involving hematopoietic cells and mice embryonic fibroblasts (MEF’s) derived from wild type (wt) mice and transgenic mice (Tg) containing high levels of mTERT expression in a large range of somatic tissues resulting in larger telomeres and increased telomerase activity not shown in this figure. The technique used to measure telmomere length was field inversion gel electrophoresis (Fig A). The telomere lengths were measured in MEF’s from both wt mice and Tg mice. Lanes 1-3 include MEF telomeres from founder line A and lanes 4-7 include telomeres from founder line B. The bands from the gel electrophoresis were taken and quantified (Fig 3B) as a mean length from the two founder lines giving wt mice telomere lengths of about 50 kb and Tg mice significantly larger (P= .018) telomere lengths measured at 70kb. This figure suggest a strong correlation between mTERT expression and telomere length (a result of telomerase activity). Therefore, Tg mice is concominant with increased telomerase activity and telomere length.
Figure 3C 3D 3E
To measure proliferation, a 3t9 protocol was used in which MEF’s were cultured for 3 days. The doubling time (proliferation rate) was then taken as a log2 value (number harvested/number plated). This part of the experiment was repeated through 19 different passages to observe proliferation rates over a range of time. Figure 3C shows doubling time over a period of time. Tg mice are represented by 12 independent MEF cultures and wt mice are represented by 13 independent cultures and both doubing time means are plotted on the graph as a black triangles (Tg) and as grey circles (wt) in relation to the passage. Both littermates exhibited a decrease in doubling time from passages 7-10 with 76% of the Tg mice exhibiting slow growth and 67% of wt mice exhibiting slow growth at the same passage. This data shows no significant difference in the rate of timing of growth arrest between each group. Ultimately, this data indicates that senescence response is not a result of telomere shortening. Additional proliferation rates were measured in which thymidine incorporation was used to determine proliferation rates. The more thymidine that is used is indicative of higher DNA synthesis. If low amounts of thymidine were used, this is indicative of low DNA synthesis suggesting low levels of proliferation. Thymidine incorporation was measured in single cell hematopoietic stem cells derived from the thymus, spleen, and bone marrow (Figure D & E). These single cells were combined to form triples in 96 well plates and different mitogen factors were administered to stimulate cell proliferation. Thymidine exhaustion was measured as a measure of proliferation and results were plotted for each well for both Tg and Wt mice as depicted in Figure 3 D & E. This data suggests that proliferation rates do not vary amongst Tg and wt mice. From this, it can be assumed that mTERT overexpression and its concomitant sideeffects do not affect Heamatopoietic stem cells responsiveness to mitogens (proliferation stimulators).
- Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4.
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- Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. ISBN:0879697164