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KEY_BIPN148_SecondMidterm_Fall2011 - BIPN148 2nd Midterm...

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BIPN148, 2nd Midterm Points:______ Fall 2011 Name:____________________________, ID:____________________ 2 | P a g e Question 1. (25 points) (A) Explain the techniques and the experimental design that resulted in each of the images below. Include drawings of the gene constructs that were used in the experiments. Mouse 1: Fos-promotor is activated in all cells and generates tTA, but doxycycline prevents activation of the second construct. Mouse 2: Most cells have low levels of neuronal activity and the Fos-promotor is thus not activated and tTA is not produces. Even though the second construct could potentially be activated, activation does not occur in any of the cells in which that lack tTA expression. Mouse 3: The seizure activates all cells, which activates the Fos-promotor and expression of tTA in most cell. The presence of tTA in the absence of doxycyline activates the expression of tTA* and tauLacZ. LacZ generates a dye when the brain tissue is stained. Mouse 4. During the seizure in the absence of doxycyline, the tTA* expression has begun. tTA* continues to activate the TetO promoter even when tTA is not produced from the Fos-promotor or when doxycyline is present to prevent binding of tTA to the TetO propmoter). tTA* thus results in self-sustaining expression of the second construct, which also expresses LacZ. When doxycyline is returned to the diet, the self-sustaining expression of LacZ results in staining of cells that were active during the earlier time period (i.e., during the seizure activity). (B) What is the advantage of this technique compared to directly labeling immediate early genes? When directly labeling immediate early genes, only neuronal activity that occurred minutes and hours before extracting the brain can be visualized. The molecular techniques described above make is possible to visualize neuronal activity that occurred days before extracting the tissue. By directly labeling immediate early genes and by using the gene constructs, two time points that are days apart can thus be compared.
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BIPN148, 2nd Midterm Points:______ Fall 2011 Name:____________________________, ID:____________________ 3 | P a g e (C) Using this technique, design an experiment that would allow you to determine whether the same hippocampal cells were active when an animal was running in the exact same 3ft by 3ft arena at two different times that are separated by 2 days. Explain the experimental design and draw a figure of the expected results. Each animal is running in the box for 10 minutes on the first and on the third day. On the first day, the animal is not on doxycycline and the self-sustaining tetO construct is thus activated in all cells that have place fields in the box (i.e., about 30 % of the cells). On the third day, the
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