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

Functional magnetic resonance imaging is a technique used to visualize not only the neural anatomical images created by traditional MRI scans, but also overlaid images of event-related hemodynamic responses in the brain. The hemodynamic activation levels refer to the amount of blood oxygenation ocurring at a particular "voxel" of the image, which is a kind of three-dimensional pixel. This hemodynamic response is often referred to as BOLD (blood-oxygen level dependent) contrast. High BOLD contrast reflects a decreased amount of deoxygenated hemoglobin present in the brain. General changes in BOLD signal are highly correlated with changes in blood flow to different regions of the brain. Images of both anatomical and functional (BOLD) data are recorded every few seconds. Data can be analyzed in such a way as to contrast the activations associated with two separate paradigms, effectively subtracting the activation of one dataset from another and presenting the difference visually. This technique is generally applied to psychophysical ventures, quantifying the results of a multitude of psychological questions.

Lecture 2 Model Systems

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

Lecture 2 Techniques

What can these be used for?

Biochemistry

Genetics: mutation and over expression

A genetic mutation is a permanent change in the DNA sequence that makes up a gene. Mutations can affect a single DNA building block or even a large segment of an entire chromosome. Mutations may be induced in an egg or sperm cell or after fertilization; these changes are termed new (de novo) mutations, and may be experimentally beneficial for studying genetic diseases or for creating transgenic animal models that mimic aspects of human disease.

The protein encoded by a particular gene may be expressed in an increased quantity ("over-expression") such that the phenotype of the organism can be significantly altered. Two commonly used techniques to create gene over-expression are to either increase the number of the copies of the gene, or, to increase the binding strength of the promotor.

Co-culture on a 3D collagen gel matrix

Antibpdy staining, also known as immunostaining, is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. The term immunostaining was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941. Now however, immunostaining encompasses a broad range of techniques used in histology, cell biology, and molecular biology that utilise antibody-based staining methods.

Cloning genes and expressing them in cell culture

Forward genetic screen

Genetic screens test and identify organisms with a specific phenotype. A forward genetic screen searches for new genes or mutant alleles, which rarely occur in nature. Hence, scientists perform a forward genetic screen by exposing the individual to a mutagen in order to induce mutations in their chromosome(s). Mutagens such as random DNA insertions by transformation or active transposons can also be used to generate new mutants.

Dye injection

Poo assay

Explant overlay assay

Incubating slices in media with chemical cues

Mammalian pyramidal neurons

Pyramidal cells are the primary projection neurons in the cerebral cortex and the hippocampus of the central nervous system (CNS, brain). Pyramidal cells have a pyramid-shaped cell a long and branching dendritic tree. An axon that carries nerve impulses emerges from one end of the cell. The axon may have local collateral branches but also project outside their region. These cells are multipolar neurons with a single apical dendrite and compose up to 80% of the neurons in the mammalian cortex. Pyramidal cells are excitatory neurons and release glutamate as their neurotransmitter.

Lecture 3 Model Systems

What are the advantages of each?

Drosophila olfactory system

Three-eye frogs

"An extra eye primordium was implanted into the forebrain region of embryonic Rana pipiens. During development both normal and supernumerary optic tracts terminated within a single, previously uninnervated tectal lobe. Autoradiographic tracing of either the normal or supernumerary eye's projection revealed distinct, eye-specific bands of radioactivity running rostrocaudally through the dually innervated tectum. Interactions among axons of retinal ganglion cells, possibly mediated through tectal neurons, must be invoked to explain this stereotyped disruption of the normally continuous retinal termination pattern." [[1]]

Lecture 3 Techniques

What can these be used for?

In vitro stripe assay

Creating a stripe assay involves affixing various substrates of interest into thin (~50 micrometers width) stripes onto a tissue-culture dish (thus, "in vitro"). One can then apply another substance to the culture dish and observe the effects of combination of both substances on the dish. For instance, one might wish to understand the molecular differences between anterior and posterior tectum to explain retinal axon patterning (this was done by Walter et al. in 1987, pg 13 of lecture 3 notes). To do this using the stripe assay, one would extract the membranes from anterior or posterior tectum and place them in alternating stripes, using flourescent labels to distinguish the two types of tissue. Then, temporal or nasal axons are allowed to grow on the stripes. Observing the results of such a test reveals that temporal retinal axons do indeed recognize the position-specific properties of the tectal cell membranes, because the temporal axons are attracted by the anterior membranes and repelled by the posterior tectal membranes. Thus, the in vitro stripe assay is a useful tool for understanding in vivo processes.

2D gel electrophoresis

A 2D gel electrophoresis is a process whereby proteins may be compared visually. The "gel" refers to a matrix of a specifically chosen polymer used to separate the molecules of analysis. "Electrophoresis" is the term that describes the electro-motive force that is used to push the molecules along the gel matrix. Molecules are applied to wells at one end of the matrix, and an electric current is applied, causing the molecules to move in a certain direction (depending on their electric charge, towards the anode if negative and towards the cathode if positive. Visualization of the progress of the molecules is made possible by dyes. The example in lecture three comes from Drescher et al. (1995): the gel electrophoresis is used to comopare proteins from anterior and posterior tectal membrane (thus, "2D"). The ligand Ephrin for the Eph receptor tyrosine kinase was found to be present in posterior, but not anterior tectal membrane. The Ephrin mRNA was revealed to be expressed in a gradient from posterior to anterior tectum.

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

Knock-out mice are genetically engineered animals with one or more genes that are made inoperable through a gene knock-out. Knock-out animals are significant to research because they allow us to test and identify the function of an identified gene whose effect is partially or fully unknown. Knock-out techniques are usually performed in mice, which are genetically similar to humans; this procedure is also easier to perform in mice compared to rats, in which knock-outs have only been possible since 2003. A typical procedure for creating knock-out mice are as follows:

1) Isolate the gene to be knocked-out from a mice genome library. A similar DNA sequence to the gene of interest is synthesized, but is made with significant changes so that the gene is inoperable. 2) Isolate stem cells from a mouse morulla, which can be grown in vitro. 3) Combine the stems cells with the re-created DNA sequence. Some of the cells will be able to incorporate the new DNA into their genomic sequence. 4) Insert stem cells into mouse blastocyst cells, then implant into a mouse uterus to complete the pregnancy. 5) Newborn mice are chimeras, sometimes not fully knocked-out mice. These animals are then crossed with other chimeras to potentially produce an offspring that is a full knock-out transgenic mouse.

Monocular enucleation

Paper 1 Model Systems

What are the advantages of each?

Chick optic tectum

Mouse superior colliculus

Mouse retina

Paper 1 Techniques

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.

Lecture 4

Bungarotoxin

Toxin harvested from the snake species Bungarus multicinctus that binds Acetylcholine receptors and therefore paralyzes its prey. Alpha bungarotoxin is used as a label for Acetylcholine receptors.

Agrin

A proteoglycan made by nerve and glia. Agrin is transported to the nerve terminal and synaptic cleft. Due to the phenotype of agrin knockout mice (dispersed acetylcholine receptors), agrin was believed to be the factor which organizes the aggregation of acetylcholine receptors into clusters. Later experiments in model systems in which agrin could not have been present due to the absence of the pre synaptic nerve (Homeobox 9 or HB9 knockouts) showed that Agrin was not necessary for clustering. It has since been elucidated that agrin stops the dispersion of acetylcholine receptors. Dispersion of acetylcholine receptors is caused by the receptor's own ligand, the neurotransmitter acetylcholine.

Proteoglycan

A class of glycoproteins which contain glycosaminoglycan chains

Glycan

The polysaccharides which form the carbohydrate moiety of glycoproteins.

MuSk

A receptor tyrosine kinase found in muscle necessary for aggregation of Acetylcholine receptors into clusters. MuSK co-localizes with Acetylcholine receptors. Its expression peaks during the formation of neuro-muscular junctions.

Rapsyn

A cytosolic protein necessary for proper Acetylcholine aggregation. During early stages of muscle development Rapsyn co-localizes with acetylcholine receptors.

ChAT

Choline Acetyl transferase. The enzyme responsible for the synthesis of Acetylcholine Acetyl-Coenzyme A and Choline.

Neuregulin

A protein which is a known ligand for the erbB type receptor tyrosine kinase.

Lecture 5

Enhancer Promoter Screen

A screen for over expression mutant phenotypes. The genotype is created through random insertion of a strong promoter into the genome.

Lethal Enhancer Screen

A screen for a second mutation that enhances a phenotype of another mutation which by itself is not lethal.

Nernst Equation

Saltatory Conduction

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