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Today’s lecture (Based on Chapter 1): 1. Basic organization of the cell 2. The static cell vs the living cell 3. Experimental approaches in cell physiology. http://www.studiodaily.com/main/technique/tprojects/6850.html. Experimental Methods Observation of response, change, adaptation - PowerPoint PPT Presentation
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Today’s lecture (Based on Chapter 1):
1. Basic organization of the cell
2. The static cell vs the living cell
3. Experimental approaches in cell physiology
http://www.studiodaily.com/main/technique/tprojects/6850.html
Experimental Methods
Observation of response, change, adaptation
→ fluorescent labelling of cellular structures
Blue = nucleus(Hoescht staining of double-stranded DNA)
Green = mitochondria (Mitotracker staining of inner membrane)
Red = actin filaments
Green = microtubules
Blue = Nucleus
Red = actin filaments
Green = microtubules
Experimental Methods
Observation of response, change, adaptation
→ fluorescent reporting of cellular events
Visualization of a calcium transient in an isolated cardiomyocyte
http://www.k2.phys.waseda.ac.jp/F1movies/F1Prop.htm
Visualizing the rotation of the ATP synthase
Experimental Methods
Observation of response, change, adaptation
→ measurement of protein levels
Western Blot
Experimental Methods
Observation of response, change, adaptation
measurement of protein levels→ Western blot→ activity measurement
Experimental Methods
Observation of response, change, adaptation
measurement of protein levels→ Western blot→ activity measurement→ immunohistochemistry
Human Papillomavirus DNA demonstrated by In Situ Hybridisation (pink) in epithelial cells identified by indirect immunofluorescence using antibody against cytokeratin (green)
Experimental Methods
Observation of response, change, adaptation
→ measurement of protein localization within the cell
Green fluorescent protein
Experimental Methods
Observation of response, change, adaptation
→ measurement of protein localization within the cell:
GFP fusion proteins
Experimental Methods
Observation of response, change, adaptation
→ measurement of mRNA levels
Northern Blot
cDNA array
Experimental Methods
Isolated cells
Isolated cardiomyocytes (rat)
Experimental methods
1.Experimental manipulation
(a) Physical/chemical
Experimental methods
1.Experimental manipulation
(a) Physical/chemical
(b) genetic
Experimental methods
1.Experimental manipulation
(a) Physical/chemical
(b) genetic- Gene knockout
Saccharomyces cerevisiae (yeast) gene knockout collection
A mouse for every gene: the NIH mouse gene knockout project
Some shocking information about our genome:
Is bigger better?
# of genes
yeast fly worm mouse human
5500
14000
20000
30000
Don’t bet on the human genome
Some shocking information about our genome:
Not at all
# of genes
yeast fly worm mouse human
5500
14000
20000
30000
30000
Experimental manipulation
1. Physical/chemical
2. Genetic- Gene ‘knockout’- Gene expression ‘knockdown’ (RNAi)
RNAi
RISC = RNA-Induced Silencing Complex (includes the protein ‘Argonaute’)
Dicer – cuts RNA into short (~20nt) sequences
Experimental manipulation
1. Physical/chemical
2. Genetic- Gene ‘knockout’- Gene expression ‘knockdown’- Overexpression / Transgenic expression
Be careful!
Other approaches to studying the molecular physiology of animal cells…
Nucleotide and protein databases
SwissProt - amino acid sequences of all known proteins
NCBI (National Center for Biotechnology Information) – nucleotide databases
Strategies for discovery (and cure) using molecular & genetic tools
Identify effected individual↓
Sequence genome↓
Identify mutation(s)
Clone mutant gene Identify mouse homologue (BLAST)↓ ↓ ↓
Express in cells Make transgenic mouse Knockout mouse gene
Observe phenotype (if any)↓
Develop fetal screening program and correct?
For Thursday:
Read Chapter 2 on protein regulation
