CSB332H1 Lecture 17: Section 3- L17
14 May 2018
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Lecture 17:
Eliminating Proliferating NPCs:
• X-irradiation
• Anti-mitotic/anti-proliferative drugs – cytosine arabinoside (AraC)
• Transgenic approaches:
o Using stem cell specific promotors (GFAP, Nestin) to overexpress HSV-1 thymidine
kinase enzyme
§ GFAP is normally considered a marker for astrocyte (found inside)
• GFAP is also a marker for immature precursor cells/stem cells
ð Studies that use ablation techniques to assess the functional significance of neurogenesis can
be difficult to interpret because it may be accompanied by inflammatory responses
o Microglial activation – because of inflammation?
Tracing Proliferating/New Neurons:
• Pseudo-neurons are going to be found migrating from the SVZ along the RMS,
often to the olfactory bulb (in rodents)
o Doublecortin (DCX) is a marker for newly born, not fully differentiated
neurons that migrate to their target, and once there complete their
differentiation to form neurons which can be stained via NeuN
ð BrdU can also be used, which will only be taken up by dividing cells
o Cells that are expressed DCX (not fully differentiated, migratory pseudo-
neuron) will be double labeled with DCX and BrdU
Experimental Overview:
• Stroke induces neurogenesis – cells will undergo proliferation to generate newly
differentiated neurons to the region of the brain that experienced damage (lesion)
o Ablation technique can be used (radiation, chemical induced, transgenic method) to
eliminate the proliferating cells
ð Transgenic approach: Thymidine Kinase expressed is under the control of DCX promotor
o TK is normally expressed in viruses – has no deleterious effects on neurons by itself
§ DCX is a more specific marker of neuronal lineage than GFAP or Nestin
because DCS is first expressed
Specifically Targeting Neuronal Cells:
• DCX promotor was used to drive the expression of reporter gene in embryonic and adult
neuronal precursor cells:
o Co-staining with neuronal markers: BIII-Tubulin (TuJ1) and MAP2
o No co-staining with: (1) astrocytes: GFAP | (2) oligodendrocytes: GalC
ð Therefore, DCX promotor line is only found in the neuronal lineage; and will only affect
neurogenesis – has no effect on astrocytes or oligodendrocytes
o Reporter gene for neuronal markers was then fused HSV-tyrosine kinase
Document Summary
Lecture 17: x-irradiation, anti-mitotic/anti-proliferative drugs cytosine arabinoside (arac, transgenic approaches, using stem cell specific promotors (gfap, nestin) to overexpress hsv-1 thymidine kinase enzyme. Gfap is normally considered a marker for astrocyte (found inside: gfap is also a marker for immature precursor cells/stem cells. Brdu can also be used, which will only be taken up by dividing cells: cells that are expressed dcx (not fully differentiated, migratory pseudo- neuron) will be double labeled with dcx and brdu. Transgenic approach: thymidine kinase expressed is under the control of dcx promotor: tk is normally expressed in viruses has no deleterious effects on neurons by itself. Dcx is a more specific marker of neuronal lineage than gfap or nestin because dcs is first expressed. Therefore, dcx promotor line is only found in the neuronal lineage; and will only affect neurogenesis has no effect on astrocytes or oligodendrocytes: reporter gene for neuronal markers was then fused hsv-tyrosine kinase.
