- 1 Lab Attendance
- 2 Lab 1 - Introduction
- 3 Lab 2 - Microscopy
- 4 Lab 3 - Fixation
- 5 Lab 4 - Immunochemistry
- 6 References
- 7 Lab 5 - Cell Knockout Methods
- Lab 1: --Z3289738 15:29, 8 March 2012 (EST)
- Lab 2: --Z3289738 14:10, 15 March 2012 (EST)
- Lab 3: --Z3289738 14:04, 22 March 2012 (EST)
- Lab 4: --Z3289738 14:51, 29 March 2012 (EST)
- Lab 5: --Z3289738 13:58, 5 April 2012 (EST)
Lab 1 - Introduction
- 'External link' has one [ ]
- 'Internal link' has two [[ ]]
Lab 2 - Microscopy
Uploading a picture onto the wiki:
- Save picture to desktop
- 'Toolbox' - 'Upload file'
- Copy and paste the title into the wiki, put [[__]]
Figure 1: Chlamydomonas cells with flagella, plasma membrane, nucleus, and chloroplast nucleoids highlighted in neon
Robinson R (2007) Centrioles Position the Nucleus and One Another. PLoS Biol 5(6): e161. doi:10.1371/journal.pbio.005016
Homework Lab 2
Identify a reference article that uses the "superresolution" microscopy technique.
Brown TA, Tkachuk AN, Shtengel G, Kopek BG, Bogenhagen DF, Hess HF, Clayton DA. (2011). Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction. Molecular Cell Biology. PMID: 22006021
What did the paper show that normal microscopy could not show?
Superresolution fluorescence microscopy exceeded previous imaging techniques by allowing scientists to see within the small and highly compartmentalised mitochondria. Using PAML and iPALM techniques, scientists were able to visualize core dimensions and relative locations of mitochondrial nucleoids, discovering that they are much larger than previously anticipated.
Lab 3 - Fixation
* Tissue preservation for slides
Homework Lab 3
Locate a current SDS for one of the fixatives described in today's lab. Identify the properties and hazards associated with that chemical.
The SDS for Chloroform can be found at the following website: http://www.jmloveridge.com/cosh/Chloroform%20Spirit.pdf
Properties and hazards associated with Chloroform
- Colourless mobile liquid
- Chloroform odour
- Miscible with water and ethanol
- Highly flammable
- Harmful if swallowed
- Harmful: danger of serious damage to health by prolonged exposure through inhalation and if swallowed.
- Limited evidence of a carcinogenic effect.
- Carcinogen Category 3
Identify 4 papers required for your group work project. Cite on the Group Project discussion page and also on your own Individual page. Add one sentence for each as too why they are relevant to your group topic.
Taketo, M Mark. "Shutting down Wnt signal-activated cancer." Nature Genetics 36. (2004): 320-22. DOI: 10.1038/ng0404-320
In this article, New evidence suggests that Wnt signaling can be suppressed or further activated by upstream signals, even though the pathway seems to be constitutively activated by downstream mutations in cancer cells.
Macdonald, Bryan. "Wnt/β-catenin Signaling: Components, Mechanisms, and Diseases." Developmental Cell 17.1 (2009): 9-26. PMID: 19619488
This article highlights some key aspects of Wnt/β-catenin signaling in human diseases including congenital malformations, cancer, and osteoporosis, and discuss potential therapeutic implications
Luu, Hue. "Wnt/β-catenin Signaling Pathway as Novel Cancer Drug Targets." Current Cancer Drug Targets 4. (2004): 653-71. Doi: 1568-0096/04
This review discusses some of the strategies that are being used or can be explored to target key components of the Wnt/β-catenin signaling pathway in rational cancer drug discover.
Giles, R., Johan, H., Clevers, H. “Cought up in a Wnt storm: Wnt signalling in cancer”. Biochimica et Biophysica Acta, (2003). 1653 1-24. http://dx.doi.org/10.1016/S0304-419X(03)00005-2
This review considers the spectra of tumors arising from active Wnt signaling and attempts to place perspective on recent data that begin to elucidate the mechanisms prompting uncontrolled cell growth following induction of Wnt signaling.
Lab 4 - Immunochemistry
A researcher has a project for a student on a newly discovered protein called by the discoverer "Musashi".
Musashi-1 and Musashi-2 are RNA-binding proteins which play a role in asymmetric cell division of ectodermal precursor cells by regulating the translation of target mRNA.  Musashi contributes to the maintenance of neural stem cells. While Musashi-1 is frequently used as a marker for proliferating neural precursor cells, it is also expressed in epithelial stem cells including intestinal and mammary gland stem cells. Database: NCBI - Musashi
Musashi antibody #2541
- Database: Cell Signaling Technology - Musashi Antibody
- Source: rabbit
- Applications: Western Blot, Immunoflourescence (IF-F) Endogenous
- Species cross reactivity: human and mouse (determined by western blot)
- Molecular weight: 30kDa
- Anti-rabbit secondary antibodies must be used to detect this antibody
- Recommended Antibody Dilution: Wesetern blotting 1:1000; Immunofluorescence (IF-F) 1:25.
- Specificity/Sensitivity: Musashi Antibody detects endogenous levels of total Musashi 1 and 2 protein.
Anti-rabit secondary antibody:
- Database: Cell Signaling Technology - Alexa Fluor® 488 Conjugate
- Alexa Fluor 488 goat anti—rabbit IgG.
- Supplied by Life Technology
- Size 0.5mL for $440.
- It is a green flourescence.
- Formulated at 2 mg/ml.
- Specificity/Sensitivity: F(ab’)2 fragments are prepared from goat antibodies that have been adsorbed against pooled human serum, mouse serum, plasmacytoma/hybridoma proteins and purified human paraproteins.
Lab 5 - Cell Knockout Methods
See: 2011 Lab 5
List the key differences in the generation of transgenic and gene knock-out mice.
- Transgenic mice are used to study overexpression of a gene product. Mice are generated by DNA microinjection of fertilized oocytes. The fertilized egg is transfered to a pseudo-pregnant female. Results in random integration of the DNA, and the gene product os over-expressed. The advantages inculde a relative high rate of insertion of the injected gene into the genome, however the random insertion can lead to position effects.
- Gene targeting is used to delete or mutate an existing cell. Knockout mice are generated by the injection into a blastocyst of genetically modified ES cells. The blastocytes are transfered to a pseudo-pregnant female. Chimeric mice are made., and the gene product is missing or mutated. The advantages include specific insertion of a gene at specific location or removal of specific genes (KO) or mimic recessive disorders (loss of function mutations). The disadvantages are the low level of ES cells with wanted gene inserted, and that further breeding is nexessary to obtain non-chimeric homozygous animal.
Describe how genetically modified mice have been used to study the biological function of tropomysin. In your answer include: i. The type of cell used ii. The measurements used to determine whether any phenotypic changes were observed iii. A model to explain how actin changes in the levels of tropomysin can lead to the observed phenotypic changes.