BIO254:Toolbox OLD

Lecture 1 Model Systems

What are the advantages of each?

Mammalian visual system

Mammalian olfactory system

Mammalian sense of smell depends on chemoreceptors. The olfactory sensors are sensory neurons embedded in a layer of epithelial tissue at the top of the nasal cavity. These neurons directly project their axons to the olfactory bulb of the brain (i.e. they are not relayed through the thalamus like other senses' axons). Their dendrites end in olfactory hairs on the surface of the nasal epithelium. Each functional olfactory receptor protein that is expressed is found in a limited number of sensory cells in the olfactory epithelium. All of the cells that express the same receptor protein project to the same regions in the olfactory bulb. A given odorant molecule may bind to one or to more than one receptor protein. Therefore each odorant molecule can excite a unique combination of cells in the olfactory bulb, so an olfactory system with even hundred of different receptor proteins can discriminate a large number of cells. Interestingly, the more odorant molecule that bind to receptors, the more action potentials are generated, and the greater the intensity of the perceived smell.

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

Caenorhabditis elegans is a model system due to its short life cycle (about four days) and ease of maintenance. These nematodes are also transparent, which allow for visual assays (such as with fluorescent proteins). In the study of neurobiology, C. elegans is useful because it has a simple nervous system of 302 neurons. For each of these neurons, the morphology and the connectivity to other neurons is known through electron micrographs.

Most sensory neurons of C. elegans can be categorized into two groups. The first type consists of chemosensory neurons with channels that are exposed to the external environment. The second group consists of mechanosensory neurons that lack these channels to the environment.

The interneurons of C. elegans vary in their function and types of connections. Some receive synaptic input from only a few neurons, while others receive signals from many neurons.

C. elegans neurons have the interesting property in which the presynapses and postsynapses are not localized to only after or before the cell body, respectively. For example, the interneuron AVE has a postsynaptic region and presynaptic region which both follow after the cell body.

Drosophila embryo

Embryonic development has been studied extensively in the Drosophila embryo, particularly the establishment of the dorsal-ventral and anterior-posterior axis, as well as segmentation in Drosophila. Patterning relies mainly on give sets of genes that create gradient and combinatorial coding to control development. These genes include maternal effect genes, gap genes, pair-rule genes, segment polarity genes, and hox genes. The body axes of the embryo are prefigured in the oocyte by maternal effect genes. These are prelocalized cytoplasmic determinants as well as localized extracellular signals (signals in the egg shell covering). Scientists have identified about 50 maternal mRNA types that build up localized determinants during oogenesis that are pre-determined before zygotic genome is turned on. More specifically, the dorsal-ventral axis is specified by an extracellular signal called spatzle. Molecules laid down in the ventral extracellular egg covering during oogenesis locally activate a ligand (spatzle). Spatzle quickly and locally binds its receptor, activating a signal transduction cascade that releases the transcription factor dorsal from a cytoplasmic inhibitor called cactus by degrading cactus. As a result, dorsal enters nuclei on the ventral side. dorsal protein is localized in the nucleus in a gradient on the ventral side of the blastoderm embryo. The gradient of activating/deactivating of transcription factor can regulate how target genes get expressed, so named because of what happens when “dorsal” is absent. The dorsal transcription factor activates the expression of target genes that specify that ventral cells become mesoderm with specifically ventral patterning. The gradient of activity of the dorsal transcription factor sets up several different domains of target gene expression. The gradient of activation of transcription factor (high ventral, low dorsal) and affinity of binding sites, determines how embryo pattern will develop based on signaling pathways. The action of the bicoid gene during oogenesis is required to set up conditions for development of anterior structures in the embryo. Bicoid mRNA, like dorsal, is a localized cytoplasmic determinant that is localized to the anterior pole of the oocyte during oogenesis (by motor proteins moving on microtubule tracks). After fertilization the mRNA is translated and bicoid protein diffuses out to form a gradient. Bicoid encodes the transcription factor that turns on target genes when the zygotic embryo’s nucleus turns on. It functions as a DNA binding protein that turns on transcription of the hunchback gene in the embryo. Hunchback, in turn, acts to pattern the embryo as a gap gene. The levels of bicoid protein sets the position of hunchback expression along the anterior-posterior axis since a threshold level of bicoid is required to turn on hunchback transcription. This threshold is set by the affinity of how tightly with which cis-regulatory sequences of the hunchback gene bind bicoid protein. patterns the embryo. Hunchback transcription is blocked in the posterior region by the nanos protein. Nanos mRNA is localized posteriorly and similarly sends out a protein gradient that opposes the bicoid protein gradient as well as the maternal Hb mRNA that is distributed uniformly throughout the cell. The gap genes, which include hunchback, giant, kruppel, and knirps, are transcription factors for segmentation. There is a tapering of the proteins in each direction from where it is expressed to create gap gene domain expression overlap, creating combinations of more than one of their protein products. The peaks overall to foster complexity since across head to tail axis there are different amounts and different locations of transcription factors, and genes sensitive to these differ in triggering influences that lead to segmented expression of genes. Pair rule gene transcription is under gap protein control. There are different DNA control elements for different stripes. For example, ftz, eve, runt, and hairy, are pair rule genes whose sequences in DNA are next to pair-rule genes that regulate the stripe expression. They are around the protein coding region in DNA segments that encode information for individual stripes and are activated by when activators such as bicoid and hunchback outweigh repressors such as giant and kruppel on the DNA sequences for a stripe’s proteins to be transcribed. Segment polarity genes, such as gooseberry, make transcription factors that control patterns in even finer stripes in response to the varying levels of maternal effect, gap, and pair-rule genes. Each stripe has its own regulatory history; a single nucleotide change in binding site for a transcription factor can alter the stripe. Combinations of transcription factors act on particular silencers/enhancers to control segmentation. The regulation of lateral segments require different combinations of transcription factors, such as Wnt and Hedgehog signals that organize the pattern of bristles within each Drosophila segment. Hedgehog and Wnt are both short range signals, but Hedgehod is a secreted protein and Wnt is a signaling pathway. Adjacent cells talking to each other for feedback to reinforce each other’s signals in positive feedback. If the Hedgehog signal fades then there’s no communication within the poles of the segments and Wnt causes bristles to form on all cells. Finally, homeotic genes (Hox genes) are single transcription factors that can affect where development occurs by conferring different fates upon repeating body segments, inducing limb growth, and organizing organ placement. This is based upon their select expression along the dorsal-ventral and anterior-posterior axis in accordance to combinatorial coding of the genes described above.

Cell culture

Small amounts of undifferentiated or single cells (normally from excised animal tissue) are placed in an artificial environment. The nutrient medium depends on the experiment being conducted, but usually the medium favors cellular growth and differentiation. By using cell cultures it is possible to pin down a cause and effect relationship between the carefully controlled culture and the development of the maturing cells. Cell cultures can be manipulated by adding chemicals, nutrients, etc. to the cellular environment to test a hypothesis or achieve desired characteristic results. Favorable qualities of cells can be precisely controlled, so that each cell is identical for the particular quality being sought, allowing for repetition within experimental methods. In the case of neuroscience, axon growth, protein secretion, receptor up/down-regulation, and neurotransmitter release can all be studied and manipulated within culture to test the effects of a wide variety of cellular environments.

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

Antibody Staining

Antibody 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.

The Poo Assay

The Poo Assay is used to assess growth cone turning responses to gradients of extracellular guidance factors. It is named after its originator, Mu-Ming Poo, who used it to demonstrate the attractive turning of a growth cone towards a gradient of netrin-1 and the repulsive turning of a growth cone away from a gradient of semaphorin 3A. Isolated growth cones are cultured in a cell-free environment in vitro and then are exposed to gradients of a potential signaling molecule. Within an hour turning of the growth cone is evident and the angle of turning can be used to gauge the strength of the molecule’s signal. Turning should not be observed when the culture medium is supplemented with an antibody against the signaling molecule of interest.

Explant overlay assay

The explant overlay assay, known more commonly as the slice overlay assay, is an in vitro assay in which neuronal explants are cultured over cortical slices. The principal use of the explant overlay assay is to characterize extracellular signaling molecules that regulate neuronal differentiation and patterning. The two methods used for this purpose before the innovation of the explant overlay assay had significant shortcomings. An in vitro assay using neuronal explants cultured on an artificial substrate was problematic because the substrate was no substitute for the actual in vivo environment in which neuronal outgrowth takes place. The limitation of the second method, an in vivo assay that involved transplanting and monitoring labeled neurons, was that the chemical environment could not be manipulated like in an in vitro assay. The explant overlay assay is able to resolve both problems, making it the most effective method for studying neuronal guidance molecules and mechanisms. Franck Polleux developed the explant overlay assay in 1998 to show that the initial growth of cortical axons toward the white matter is regulated by a semaphorin signal that is expressed in the marginal zone.

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

The Drosophila olfactory system is a great model system for understanding how precise connections are made, what are the genes important for the formation of precise connections, and how formation of these precise connections are relevant for encoding olfactory information. Olfactory sensory neurons project their axons to discrete circular centers called glomeruli. At these glomeruli they connect with the dendrites of second order neurons, projection neurons. The projection neurons then send axons to the mushroom body calyx and the lateral horn for higher processing of olfactory information. The power of genetics has allowed scientists to label projection neurons. Since the advent of MARCM (Mosaic Analysis with a Repressible Cell Marker) one can label a subset of these projection neurons. One can even label a single projection neuron. Using MARCM, studies have shown that lineage and birth timing of projection neurons is correlated with their glomerular projections. MARCM has also been used to study the branching patterns of individual classes of projection neurons and the genes involved in the precise projections to single glomeruli (e.g. Sema1a, N-cadherin, Dscam).

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." ("Eye-specific termination bands in tecta of three-eyed frogs" [1])

Frogs do not have binocular vision because the outputs of the left and right eye do not converge. All retinal ganglion cells (RGCs; the cells that relay information from eye to the next level of information processing) from the left eye project their axons to the optic tectum on the right side. All RGCs from the right eye project their axons to the optic tectum on the left size. Because the left and right eyes are completely segregated there is no competition during development and no stripe formation is seen. However, when you transplant a third eye, you induce competition among axons projecting to the optic tectum. The competion between RGC axons from the transplanted and non-transplanted eyes to the same optic tectum gives rise stripes.

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

In humans, tranplanted organs are used to replace a failing or damaged organ with a working organ from a donor. In research, transplatation is useful for exploring interactions between individual organisms--for example, the unique responses of an organism's immune system or the three-eyed frog to study axonal competition during neuron growth. Several types of transplatations are done:

1) Allografts = transplanting organs or tissues from a genetically non-identical member within the same species; 2) Autografts = transplanting tissue from one area of one's body to another, usually with surplus tissue to replace damaged areas; 3) Xenografts = transplanting organs or tissues across species (example, pig's heart to human body); 4) Isografts = transplanting organs or tissues to a genetically identical member of the same species (such as a twin). This type of transplantation may overcome difficulties associated with organ rejection or triggering a recipient's immune system.

Radiolabel injection

Using radiolabeled injections, neurobiologists are able to observe cellular mechanisms and metabolisms in real-time, such as the influx and efflux of calcium within a cell. This technique is completed by making and attaching a radiolabeled tag to the compound of interest, then injecting this compound into the organism or cell system under study. Through neuroimaging techniques such as MRI, fMRI and PET, we are able to see the brain regions where certain chemicals are taken up and metabolized.

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?

HEK293 cells

HEK 293 cells are an epithelial cell line originally derived from embryonic human kidney. As an experimentally transformed cell line, HEK cells are not a particularly good model for normal cells, cancer cells, or any other kind of cell that is a fundamental object of research. However, they are extremely easy to work with, being straightforward to culture and to transfect, and so can be used in experiments in which the behavior of the cell itself is not of interest. Typically, these experiments involve transfecting in a gene (or combination of genes) of interest, and then analyzing the expressed protein; essentially, the cell is used simply as a test tube with a membrane.

Examples of such experiments include:

-A study of the effects of a drug on sodium channels
-Testing of an inducible RNA interference system
-Testing of an isoform-selective protein kinase C agonist
-Investigation of the interaction between two proteins
-Analysis of a nuclear export signal in a protein

In the Schmitt et al (2006), HEK 293 cells were used in a preliminary test to determine whether Wnt3 can regulate the growth of RGC axons. Schmitt el al created HEK293 cells transfected with the wnt3 gene in order to have the Wnt3 protein expressed in membrane fractions of HEK293 cells (Wnt3 is highly hydrophobic and associates tightly with cell membranes). They found that Wnt3-transfected HEK293 cell membranes inhibited the growth of both dorsal and ventral mouse RGC axons at higher concentrations, and stimulated the growth of dorsal but not ventral RGC axons at lower concentrations (data was not shown).

SF9 cells

An insect cell line used for the production of recombinant protein. The Sf9 cell line is derived from pupal ovarian tissue of the Fall armyworm Spodoptera frugiperda. The Sf9 cell line is highly susceptible to infection with Autographa california nuclear polyhedrosis virus (AcNPV baculovirus), and can be used with all baculovirus expression vectors. Sf9 cells are commonly used to isolate and propagate recombinant baculoviral stocks and to produce recombinant proteins. In the Schmitt et al. paper, Sf9 cells were used to overexpress Wnt3 (using the Baculovirus system) to obtain sufficient and consistent amounts of Wnt3.

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

A dominant-negative is a mutation whose gene product adversely affects the normal, wild-type gene product within the same cell. This usually occurs if the product can still interact with the same elements as the wild-type product, but block some aspect of its function.

Examples: 1. A mutation in a transcription factor that removes the activation domain, but still contains the DNA binding domain. This product can then block the wild-type transcription factor from binding the DNA site leading to reduced levels of gene activation. 2. A protein that is functional as a dimer. A mutation that removes the functional domain, but retains the dimerization domain would cause a dominate negative phenotype, because some fraction of protein dimers would be missing one of the functional domains.

The Shmitt et al Ryk dominant negative: Wnt3 knockout mice fail in early embryonic patterning because Wnt3 is important for early nervous system development. This makes it impossible to examine the function of the Wnt3 gradient on the medial-lateral axis of the mouse superior colliculus because knockout Wnt3 mice die at birth and axon termination zones form at postnatal day 8. To circumvent this difficulty, Schmitt et al generated a dominant-negative form of Ryk. This truncated Ryk protein only contained Ryk ectodomain (extracellular) and the transmembrane domain, missing the intracellular domain. This dominant negative Ryk allowed Schmitt et al to test in vivo whether blocking Wnt3-Ryk function will shift the termination zone of RGX neurons in the superior colliculus of mice medially.

In ovo electroporation

DAPI staining

Fluorescently labels cell nuclei by binding to DNA.

AP (alkaline phosphatase)

Tagged proteins

Protein overexpression

Selective gene amplification by a cell results in more templates for transcription, which is the basis of natural protein overexpression. The cell can make more of a certain gene product by increasing the number of copies of the appropriate gene and transcribing them all. This strategy takes advantage of the transcription mechanisms already in place within the cell and merely feeds them more material to transcribe. In the lab, the polymerase chain reaction (PCR) technique makes multiple copies of a DNA sequence by copying a short region of DNA many times in a test tube. It is a cyclic process in which a sequence of steps is repeated over and over: A DNA molecule with a target sequence to be copied is heated to denature it. When the mixture cools, short, artificially synthesized primers bond to the single-stranded DNA. Then dNTPs (four deoxyribonucleotide triphosphates dATP, dGTP, dCTP, and dTTP) and DNA polymerase are added to synthesize two new strands of DNA. The process is repeated, doubling the amount of DNA. One goal of recombinant DNA technology is to produce many copies (clones) of a particular gene either for the purposes of analysis or to produce its protein product in quantity. If the recombinant DNA is to make its protein it must first be transfected into a host cell. Once the host species is selected, the recombinant DNA is brought together with a population of host cells and, under specific conditions, enters some of them. One common method of identifying cells with recombinant DNA is to tag the inserted sequence with reporter genes whose phenotypes are easily observed. These phenotypes serve as genetic markers for the sequence of interest. Scientists normally use bacteria as hosts because they are easily grown and manipulated. Bacteria also contain plasmids, small circular chromosomes, which can be manipulated to carry recombinant DNA into the cell. But bacterium is not ideal for studying and expressing eukaryotic genes because they lack the splicing machinery to excise introns from the initial RNA transcript of eukaryotic genes. Many eukaryotic proteins are extensively modified after translation by reactions such as glycosylation and phosphorylation. Often these modifications are essential for the protein’s activity. So scientists use vectors rather than bacteria to carry the new DNA into host cells. Vectors already have a built-in origin of replication, a specific sequence that DNA polymerase binds to when replicating the DNA. The new DNA has to become part of a segment of DNA that contains an origin of replication (i.e. join a replication unit) in order to be replicated in the host cell as it divides. Plasmids are often used as vectors because they are small, naturally occurring in bacteria, often have only a single recognition site for a given restriction enzyme, and allows for the insertion of DNA at only one location. When the plasmid is cut with a restriction enzyme, it is transformed into a linear molecule with sticky ends that can pair with the sticky ends of another DNA fragment cut with the same restriction enzyme. Viruses can also be used as vectors to insert large numbers of base pairs into a genome. Even if the genes that cause the host cell to die and lyse are gone, the virus can still attach to a host cell and inject its DNA, which in our case is the new DNA to be expressed. Finally, expression vectors allow foreign genes to be expressed in host cells and can turns cells into protein factories, contributing to protein overexpression. Expression vectors contain the sequences for promotion, termination, and ribosome binding, which are necessary for protein synthesis in a foreign cell. Transfected eukaryotic genes are put into an expression vector which includes the appropriate sequences for transcription, translation, and all the other extra sequences needed for the protein’s expression, such as a poly-A tail, transcription factor binding sites, and enhancers. The protein to be overexpressed is inserted at the restriction site, the bacteria of choice is transfected with the expression vector, and the protein is synthesized because of its locale in the DNA. Inducible promoters that respond to a specific signal, thus initiating protein synthesis, can be inserted into the expression vector so that the production of the target protein can be controlled.

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.

Lecture 6

Shibire

Kiss and Run

Ionotropic Receptors

Metabotropic Receptors

Acetylcholine Receptors

Electron Microscopy

Current Voltage Relationship of Na+ Channels

NMDA Receptor

GABAa Receptor

MK801

APV (AP-5)

Hippocampus

SNAREs

Lecture 7

cAMP

G-proteins=

GPCRs

Narcoleptic dogs

Adenylate cyclase

IP3 and DAG

Fluorescent Proteins (e.g. GFP)

FRET Imaging

TRP Channels

CREB

EF Hands and C2 Domains

CaM Kinase II

See BIO254:CaMKII

Lecture 8

Eye Anatomy

Fovea

Rods vs. Cones

Isolated Retinal Rod Cell

Photoreceptors vs. Neurons

11 cis-retinal

Rhodopsin

Phototransduction

See BIO254:Phototransduction

cGMP gated channel

Adaptation

See BIO254:Adaptation

RGS9

Lecture 9

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25 April 2024

			00:22		The paper that launched microfluidics - Xi Ning‎‎ 7 changes history +4,723‎ [Xning098‎ (7×)]
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					00:08 (cur | prev) +4‎ Xning098 talk contribs (→‎Significance)
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24 April 2024

			18:35		User:Yanbin Huang‎‎ 2 changes history +25‎ [Yanbin Huang‎ (2×)]
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			17:49		Hu‎‎ 2 changes history +28‎ [Hugangqing‎ (2×)]
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08:02

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23 April 2024

			11:58		BioMicroCenter:People‎‎ 2 changes history +30‎ [Lttran‎ (2×)]
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11:46

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22 April 2024