BIO254:Toolbox OLD

Lecture 1 Model Systems

What are the advantages of each?

Mammalian visual system

Mammalian olfactory system

Spinal cord motor neurons

Human brain

Lecture 1 Techniques

What can these be used for?

Golgi staining

Also called the Black Reaction, Golgi staining stains a subset of cells within the brain, because staining all neurons and cellular processes would make anatomical analyses difficult and cumbersome. While the exact mechanisms behind the Golgi stain is not well understood, this technique labels axons, dendrites, and cell somas in black and brown along their entire length. Hence, neural ciruits can be visualized, tracked, and mapped. Golgi stains are made by injection of potassiumdichromate and silver nitrate; the brown-black color of neurons stems from the microcrystallization of silver chromate.

Tissue culture

Tissue cultures allow researchers to grow tissues and/or cells outside of the organism under investigation. Primary cell cultures usually have a finite life span in culture compared to cell lines which are abnormal or transformed cell lines. The availability of tissue cultures enable the study of cells in a controlled environment without the external influences found in the organisms' physiological environment. Advantages of such a technique include the ability to study specific cellular mechanisms alone, and the opportunity to manipulate cell lines to better understand developmental abnormalities.

Electron microscopy

Through the use of electrons to create an image of the object, electron microscopy provides higher magnification and superior resolving power than a light microscope by almost a magnitude of two million. Various electron microscopy techniques exist for exploring morphology and mechanisms: scanning electron microscopes give a 3D image of the sample; transmission electrion microscopes produce 2D images at impressive magnifications (up to 500 million times); and scanning tunneling microscopes determine the height of the sample surface.

Biolistic transfection (gene gun)

This technique injects cells with a heavy metal coated with plasmid DNA, and is capable of transforming almost all types of cells including their genetic information and cellular organelles. Gene guns are also effective in delivering DNA vaccines to mammals for therapy.

Genetic labeling

Patch clamp

The patch clamp method allowed detailed understanding of the action potential after it was invented by Kenneth Cole in the 1940s. This method enables us to measure the membrane potential, or voltage, at any level desired by the experimenter through use of a microelectrode placed inside the cell. The voltage clamp technique reveals how membrane potential influences ionic current flow across the membrane, and was instrumental in providing Hodgkin and Huxley with information leading to membrane ion gradients and the action potential.

Electrical stimulation

fMRI

Lecture 2

Model Systems Introduced: What are the advantages of each?
Frog visual system
Vertebrate spinal cord
C. elegans sensory and motor neurons
Drosophila embryo
Cell culture
Grasshopper
Xenopus axons in culture

Techniques and Tools Introduced: What can these be used for?
Biochemistry
Genetics: mutation and over expression
Co-culture on a 3D collagen gel matrix
Antibody staining
Cloning genes and expressing them in cell culture
Forward genetic screen
Dye injection
Poo assay
Explant overlay assay
Incubating slices in media with chemical cues
Mammalian pyramidal neurons

Lecture 3

Model Systems Introduced: What are the advantages of each?
Drosophila olfactory system
Three-eye frogs

Techniques and Tools Introduced: What can these be used for?
In vitro stripe assay
2D gel electrophoresis
Transplantation
Radiolabel injection

TTX

Tetrodotoxin. A toxin from the puffer fish that blocks voltage gated sodium channels.

TEA

Tetraethylammonium. A compound which selectively blocks voltage gated potassium channels.

Differential Display

A technique used to determine the differences in expression of mRNA between two cells under different conditions or between two different cell, using mRNA probes. This technique is rapidly being replaced by expression profiles using microarrays.

In-situ hybridization

In-situ uses mRNA probes (also called oligos) that anneal to the mRNA strand of interest in fixed animal tissue. Because the probes are usually fluorescently-tagged, this technique allows visualization of mRNA in cells/tissue, providing quantitative data on the amount of genetic information being expressed.

Knockout mice
Monocular enucleation

Paper 1

Model Systems Introduced: What are the advantages of each?
Chick optic tectum
Mouse superior colliculus
Mouse retina

Techniques and Tools Introduced: What can these be used for?
In-situ hybridization – sense controls
HEK293 cells

SF9 cells

An insect cell line (from a kind of caterpillar) used for the production of recombinant protein.

Baculovirus system

Baculovirus is a natural pathogen of the caterpillars producing the SF9 cell line. In the lab, genes are encoded into a baculovirus vector which is then used to infect SF9 cells.

Affinity-purified protein

A protein purified by passing a solution of protein through a column where the protein becomes associated with a matrix of immobilized ligand somehow attatched to the column. In most cases the protein must be tagged, or appended to a functional motif called a fusion tag. Common fusion tag-ligand pairs include: Histidine tag (6 or more extra Histidines) and the "ligands" Chelated Nickel or Cobalt, Maltose Binding Protein and its ligand dextrin, Glutathione S-transferase and its ligand reduced glutathione, and Green Fluorescent Protein and Anti-GFP antibody.

Mock infection
Blocking with antibodies or proteins
Western Blot, α-tubulin
Retina explant assay
Electroporation into ventricular zone
Dominant-negative
In ovo electroporation

DAPI staining

Fluorescently labels cell nuclei by binding to DNA.

AP (alkaline phosphatase)
Tagged proteins
Protein overexpression

sFRP2

secreted frizzled related protein 2 is an antagonist of the Wnt ligand in Wnt-Frizzled mediated cell signalling.

DiI

A lipophillic compound used to label cells. DiI has affinity for any cell membrane and is therefore not cell specific, but will only label the cell individually injected with DiI.

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4 May 2024

18:10

The Paper that Launched Microfluidics - Xi Ning‎ diffhist −86‎ Xning098 talk contribs (→‎Electrokinetic effect)

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3 May 2024

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2 May 2024