Lab Manual for Biomedical Research

Chromosome In Situ Hybridization

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A modern approach to the specific location of genes on chromosomes is a technique for the hybridization of DNA and RNA "in situ." With this procedure, specific radioactive RNA or DNA (known as probes) can be isolated (or synthesized "in vitro") and then annealed to chromosomes which have been treated in such a manner that their basic double stranded DNA has been "melted" or dissociated.

In theory, and fortunately in practice, when the DNA is allowed to re-anneal, the probe competes for the binding, but only where it mirrors a complimentary sequence. Thus, RNA will attach to the location on the chromosome where the code for its production is to be found. DNA will anneal to either RNA which is still attached to a chromosome, or to the complimentary sequence DNA strand within the chromosome. Since the probe is radioactive, it can be localized via autoradiographic techniques.

Finally, it is possible to produce an RNA probe that is synthesized directly from repetitive sequences of DNA, such as that found within the nucleolar organizer region of the genome. This RNA is known as cRNA (for copied RNA) and is a convenient source of a probe for localizing the nucleolar organizer gene within the nucleus, or on a specific chromosome.

The use of in situ hybridization begins with good cytological preparations of the cells to be studied, and the preparation of pure radioactive probes for the analysis. The details depend upon whether the hybridization is between DNA (probe) and DNA (chromosome), DNA (probe) and RNA (chromosome), or between RNA (probe) and DNA (chromosome).

Preparation of the Probe:

Produce radioactive RNA by incubating the cells to be measured in the presence of H-uracil, a specific precursor to RNA. Subsequent to this incubation, extract rRNA from the sample and purify through differential centrifugation, column chromatography or electrophoresis. Dissolve the radioactive RNA probe in 4X Saline-Citrate containing 50% formamide to yield a sample that has 50,000 to 100,000 counts per minute, per 30 microliter sample, as determined with a scintillation counter. Add the formamide is added to prevent the aggregation of RNA.

Preparation of the Slides:

Fix the materials to be studied in either 95% ethanol or in 3:1 methanol:water, attach to pre-subbed slides (as squashes for chromosomes) and air dry.

Hybridization

Place the air dried slides into a moist chamber, usually a disposable petri dish containing filter paper and carefully place 30 microliters of RNA probe in 4X SSC-50% formamide onto the sample.

Carefully add a cover slip (as in the preparation of a wet mount), place the top on the container and place in an incubator at 37° C for 6-12 hours.

Washing:

Pick up the slides and dip into 2X SSC so that the coverglass falls off.

Place the slides in a coplin jar containing 2X SSC for 15 minutes at room temperature.

Transfer the slides to a treatment with RNase (50 microgram/ml RNase A, 100 units/ml RNase T1 in 2X SSC) at 37° C for 1 hour.

Wash twice in 2X SSC, 15 minutes each.

Wash twice in 70% ethanol, twice in 95% ethanol and air dry.

Autoradiography:

Add photographic emulsions to the slides and after a suitable exposure period, develop the slides, counterstain and add cover slips.

Analyze the slides by determining the location of the radioactive probe on the chromosomes or within the nuclei.

(Dr. William H. Heidcamp)

H and E Stain Full Protocol

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Paraffin processing of tissue

Fixation of Tissues

1. Where the best possible morphology is required, animals should be anesthesized and subjected to cardiac perfusion with saline, followed by a 10% formalin flush. If biochemical studies need to be performed on the tissue, a 10% formalin flush should not be used as it may interfere with subsequent analysis.

2. For routine stains where perfusion is not required, tissue is sectioned and drop-fixed in a 10% formalin solution. Fixative volume should be 20 times that of tissue on a weight per volume; use 2 ml of formalin per 100 mg of tissue.

3. Due to the slow rate of diffusion of formalin (0.5 mm hr), tissue should be sectioned into 3 mm slices on cooled brain before transfer into formalin. This will ensure the best possible preservation of tissue and offers rapid uniform penetration and fixation of tissue within 3 hours.

4. Tissue should be fixed for a minimum 48 hours at room temperature.

5. After 48 hours of fixation, move tissue into 70% ethanol for long term storage.

6. Keep fixation conditions standard for a particular study in order to minimize variability. (Although set times are best, tissue may be fixed for substantially longer periods without apparent harm.

A few notes on fixation

The usual fixative for paraffin embedded tissues is neutral buffered formalin (NBF). This is equivalent to 4% paraformaldehyde in a buffered solution plus a preservative (methanol) which prevents the conversion of formaldehyde to formic acid. Because of the preservative, NBF has a shelf life of months, whereas 4% PF must be made fresh. Optimal histology requires adequate fixation, about 48 hrs at room temperature for thinly sliced tissues. Inadequately fixed tissues will become dehydrated during tissue processing, resulting in hard and brittle specimens. Alcohol based fixatives generally do not give good morphology but may be useful in special cases (such as BrdU staining). A particular challenge for the histopathology is immunostaining fixed specimens. In many cases formaldehyde fixation will prevent recognition of epitopes by the primary antibody. Occasionally, “antigen retrieval” procedures will improve results but usually frozen sections are a better bet. An alternative approach, suitable for thin or porous tissues, is to perform immunohistochemistry on fresh tissues and then post-fix and embed the tissues in paraffin.

Decalcification of bone (optional):

After fixation, bone,must be decalcified, or else it won’t cut on the microtome:

Immerse tissue cassette in 11% formic acid with a stir bar overnight in a fume hood.

Rinse in running water for 30- 60 minutes (the smell should be gone).

Storage in 70% Ethanol:
After adequate fixation tissues are transferred to 70% ethanol and may be stored at 4°C.

Paraffin infiltration

In this procedure, tissue is dehydrated through a series of graded ethanol baths to displace the water, and then infiltrated with wax. The infiltrated tissues are then embedded into wax blocks. Once the tissue is embedded, it is stable for many years.

The most commonly used waxes for infiltration are the commercial paraffin waxes. A paraffin max is usually a mixture of straight chain or n-alkanes with a carbon chain length of between 20 and 40; the wax is a solid at room temperature but melts at temperatures up to about 65°C or 70°C. Paraffin wax can be purchased with melting points at different temperatures, the most common for histological use being about 56°C–58°C, At its melting point it tends to be slightly viscous, but this decreases as the temperature is increased. The traditional advice with paraffin wax is to use this about 2°C above its melting point. To decrease viscosity and improve infiltration of the tissue, technologists often increase the temperature to above 60°C or 65°C in practice to decrease viscosity.

In the schedule below, it is presumed that the working day is from 8:00 a.m. to 5:00 p.m. If other than that, appropriate adjustments should be made.

Tissue preparation
Thickness	No more than 3 mm thick.
Area	20 mm × 30 mm.
Fixed tissue	Cut large organs into 3 mm slices and store in neutral buffered formalin for 48 hours. Select tissue from fixed areas, trim to size and refix until the evening. If the trimmed sample is visibly unfixed, refix for a further 24 hours.
Unfixed tissue	Slices of tissue should be thoroughly fixed before processing.
Times	All times in processing fluids for this schedule are for tissues 3 mm thick or less. Tissues thicker than that will require longer times.
Clearing agent	Xylene or another clearing agent that will clear tissues in similar times should be used.
Processing time	This schedule takes 12 hours, and processes overnight. On weekends tissues should be left in fixative until Sunday evening with a 48 hour delay.

Trim fixed tissues and keep in neutral buffered formalin (NBF) until ready to proceed. Put tissues in a labeled (usually with pencil, as solvents dissolve the ink) cassette.

Once fixed, tissue is processed as follows, using gentle agitation, usually on a tissue processor, as follows:

1. 70% ethanol for 1 hour.

2. 95% ethanol (95% ethanol/5% methanol) for 1 hour.

3. First absolute ethanol for 1 hour .

4. Second absolute ethanol 1½ hours .

5. Third absolute ethanol 1½ hours.

6. Fourth absolute ethanol 2 hour.

7. First clearing agent ( Xylene or substitute) 1 hour.

8. Second First clearing agent (Xylene or substitute) 1 hour.

9. First wax (Paraplast X-tra) at 58°C for 1 hour.

10. Second wax (Paraplast X-tra) at 58°C 1 hour.

Due to the viscosity of molten paraffin wax, some form of gentle agitation is highly desirable. If the processor is to be run overnight it should be programmed to hold on the first ethanol bath and not finish until the next morning so the specimens do not sit in hot paraffin longer than the time indicated. If specimens are fresh they may incubate in formalin in the first stage on the machine. It is important to not keep the tissues in hot paraffin too long or else they become hard and brittle. Processed tissues can be stored in the cassettes at room temperature indefinitely.

Embedding tissues in paraffin blocks

Tissues processed into paraffin will have wax in the cassettes; in order to create smooth wax blocks, the wax first needs to be melted away placing the entire cassette in 58°C paraffin bath for 15 minutes. Turn the heat block on to melt the paraffin one hour before adding the tissue cassettes.

1. Open cassette to view tissue sample and choose a mold that best corresponds to the size of the tissue. A margin of at least 2 mm of paraffin surrounding all sides of the tissue gives best cutting support. Discard cassette lid.

2. Put small amount of molten paraffin in mold, dispensing from paraffin reservoir.

3. Using warm forceps, transfer tissue into mold, placing cut side down, as it was placed in the cassette.

4. Transfer mold to cold plate, and gently press tissue flat. Paraffin will solidify in a thin layer which holds the tissue in position.

5. When the tissue is in the desired orientation add the labeled tissue cassette on top of the mold as a backing. Press firmly.

6. Hot paraffin is added to the mold from the paraffin dispenser. Be sure there is enough paraffin to cover the face of the plastic cassette.

7. If necessary, fill cassette with paraffin while cooling, keeping the mold full until solid.

8. Paraffin should solidify in 30 minutes. When the wax is completely cooled and hardened (30 minutes) the paraffin block can be easily popped out of the mold; the wax blocks should not stick. If the wax cracks or the tissues are not aligned well, simply melt them again and start over.

The tissue and paraffin attached to the cassette has formed a block, which is ready for sectioning.Tissue blocks can be stored at room temperature for years.

Sectioning tissues

Tissues are sectioned using a microtome. Turn on the water bath and check that the temp is 35-37ºC. Use fresh deionized water (DEPC treated water must be used if in situ hybridization will be performed on the sections). Blocks to be sectioned are placed face down on an ice block or heat sink for 10 minutes. Place a fresh blade on the microtome; blades may be used to section up to 10 blocks, but replace if sectioning becomes problematic. Insert the block into the microtome chuck so the wax block faces the blade and is aligned in the vertical plane.

Set the dial to cut 10 µM sections to order to plane the block; once it is cutting smoothly, set to 5 µM
sections . The blade should angled at 5º. Face the block by cutting it down to the desired tissue plane and discard the paraffin ribbon. If the block is ribboning well then cut another four sections and pick them up with forceps or a fine paint brush and float them on the surface of the 37ºC water bath. Float the sections onto the surface of clean glass slides. If the block is not ribboning well then place it back on the ice block to cool off firm up the wax. If the specimens fragment when placed on the water bath then it may be too hot.

Place the slides with paraffin sections on the warming block in a 65°C oven for 20 minutes (so the wax just starts to melt) to bond the tissue to the glass. Slides can be stored overnight at room temperature.

Haematoxylin Eosin (H&E) staining

Lung tissue stained with the H&E technique. Nuclei are darkly stained in this image.

H&E stain, HE stain or hematoxylin and eosin stain, is a popular staining method in histology. It is the most widely used stain in medical diagnosis; for example when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E and termed H&E section,H+E section, or HE section.

The staining method involves application ofhemalum, which is a complex formed from aluminium ions and oxidized hematoxylin. This colors nuclei of cells (and a few other objects, such as keratohyalin granules) blue. Materials colored blue by hemalum are often said to be basophilic, but this is an incorrect use of the word. The nuclear staining is folowed by counterstaining with an aqueous or alcoholic solution of eosin Y, which colors eosinophilic other structures in various shades of red, pink and orange.

Solutions:

Haematoxylin Solutions

Haematoxylin stains are commonly employed for histologic studies, often employed to color the nuclei of cells (and a few other objects, such as keratohyalin granules) blue. The mordants used to demonstrate nuclear and cytoplasmic structures are alum and iron, forming lakes or colored complexes (dye-mordant-tissue complexes), the color of which will depend on the salt used. Aluminium salt lakes are usually colored blue white while ferric salt lakes are colored blue-black.

The three main alum haematoxylin solutions employed are Ehrlich’s haematoxylin, Harris’s haematoxylin and Mayer’s haematoxylin. The name haemalum is preferable to “haematoxylin” for these solutions because haematein, a product of oxidation of haematoxylin, is the compound that combines with aluminium ions to form the active dye-metal complex. Alum haematoxylin solutions impart to the nuclei of cells a light transparent red stain which rapidly turns blue on exposure to any neutral or alkaline liquid.

Alum or potassium aluminium sulfate used as the mordant usually dissociates in an alkaline solution, combining with OH? of water to form insoluble aluminium hydroxide. In the presence of excess acid, aluminium hydroxide cannot be formed thus failure of aluminium haematoxylin dye-lake to form, due to lack of OH? ions. Hence, acid solutions of alum haematoxylin become red. During staining alum haematoxylin stained sections are usually passed on to a neutral or alkaline solution (e.g. hard tap water or 1% ammonium hydroxide) in order to neutralize the acid and form an insoluble blue aluminium haematin complex. This procedure is known as blueing.

When tap water is not sufficiently alkaline, or is even acid and is unsatisfactory for blueing haematoxylin, a tap water substitute consisting of 3.5 g NaHCO3 and 20 g MgSO4.7H2O in one liter of water with thymol (to inhibit formation of moulds), is used to accelerate blueing of thin paraffin sections. Addition of a trace of any alkali to tap or distilled water also provides an effective blueing solution; a few drops of strong ammonium hydroxide or of saturated aqueous lithium carbonate, added immediately before use, are sufficient for a 400 ml staining dish full of water. Use of very cold water slows down the blueing process, whereas warming accelerates it. In fact, the use of water below 10°C for blueing sections may even produce pink artifact discolorations in the tissue.

The staining of nuclei by hemalum does not require the presence of DNA and is probably due to binding of the dye-metal complex to arginine-rich basic nucleoproteins such as histones. The mechanism is different from that of nuclear staining by basic (cationic) dyes such as thionine or toluidine blue. Staining by basic dyes is prevented by chemical or enzymatic extraction of nucleic acids. Such extractions do not prevent staining of nuclei by hemalum.

Eosin Solutions

Eosin is a fluorescent red dye resulting from the action of bromine on fluorescein. It can be used to stain cytoplasm, collagen and muscle fibers for examination under the microscope. Structures that stain readily with eosin are termed eosinophilic.Eosin is most often used as a counterstain to haematoxylin in H&E (haematoxylin and eosin) staining. Eosin stains red blood cells intensely red. Eosin is an acidic dye and shows up in the basic parts of the cell, ie the cytoplasm. For staining, eosin Y is typically used in concentrations of 1 to 5 percent weight by volume, dissolved in water or ethanol. For prevention of mold growth in aqueous solutions, thymol is sometimes added. A small concentration (0.5 percent) of acetic acid usually gives a deeper red stain to the tissue.

Other colors, e.g. yellow and brown, can be present in the sample; they are caused by intrinsic pigments, e.g. melanin.

Some structures do not stain well. Basal laminae need to be stained by PAS stain or some silver stains in order to exhibit appropriate contrast. Reticular fibers also require silver stain. Hydrophobic structures also tend to remain clear; these are usually rich in fats, eg. adipocytes, myelin around neuron axons, and Golgi apparatus membranes.

Phosphate Assay – Lipids

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Protocol

Introduction: This can be used for determining the phospholipids content. Be careful when

adding acid to the tubes. Most work should be done in the hood. It is best to practice using old

samples and a standard curve before using important experimental samples.

Protocol: Clin Chem Acta 121, 111-116. 1982

1 - Prepare a standard curve phosphate curve in triplicate.

(0, 25, 50, 75, 100, 150, 200, 400 μl of 1 mM KH2PO4) – calculate how many μg of phosphate are in each

tube you will need this later.

2 - Add 25, μl of sample in separate tubes. Do in triplicate.

3 - Dry standards in heating block inside hood

- dry organic solvent (lipid samples) under nitrogen.

4 - Add 100 μl concentrated H2SO4 to all tubes.

5 - Vortex and put tubes in the heating block for 10 min.

6 - Allow tubes to cool to room temperature and add 50 μl of 6% hydrogen peroxide

7 - Vortex tubes and place in heating block for 40 minutes.

8 - Allow tubes to cool and add 2 ml of H2O, mix well.

9 - Add 800 μl of Color reagent to each tube.

10 - Boil samples on hot plate or heat block for 10 - 15 minutes or until highest standard turns a very dark blue.

11 - Transfer to 1 ml cuvettes and read absorbance at 797 nm

12 - Plot standards as absorbance vs. μg phosphate

Solutions

50 1 mM KH2PO4

Color reagent

1:1 ammonium anhydride molybdic acid (0.625g/50ml): ascorbic acid (0.45 g / 50 ml)

Mix just prior to use.

6% H2O2 - (1 ml of 30% H2O2 and 4 ml of H2O) Make just prior to use

http://www.mnstate.edu/provost/PhosphateAssayprotocol.pdf

Wallert and Provost Lab

Protein targeting (2)

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Sorting of proteins to both chloroplasts and mitochondria

Many proteins are needed in both mitochondria and chloroplasts. In general the targeting peptide is of intermediate character to the two specific ones. The targeting peptides of these proteins have a high content of basic and hydrophobic amino acids, a low content of negatively charged amino acids. They have a lower content of alanine and a higher content of leucine and phenylalanine. The dual targeted proteins have a more hydrophobic targeting peptide than both mitochondrial and chloroplastic ones.

Sorting of proteins to peroxisomes

All peroxisomal proteins are encoded by nuclear genes.

To date there are two types of known Peroxisome Targeting Signals (PTS):

Peroxisome targeting signal 1 (PTS1): a C-terminal tripeptide with a consensus sequence (S/A/C)-(K/R/H)-(L/A). The most common PTS1 is serine-lysine-leucine (SKL). Most peroxisomal matrix proteins possess a PTS1 type signal.

Peroxisome targeting signal 2 (PTS2): a nonapeptide located near the N-terminus with a consensus sequence (R/K)-(L/V/I)-XXXXX-(H/Q)-(L/A/F) (where X can be any amino acid).

There are also proteins that possess neither of these signals. Their transport may be based on a so-called "piggy-back" mechanism: such proteins associate with PTS1-possessing matrix proteins and are translocated into the peroxisomal matrix together with them.

Diseases

Peroxisomal protein tran

sport is defective in the following genetic diseases: Zellweger syndrome. Adrenoleukodystrophy (AL

D).

Refsum disease

Receptor-mediated endocytosis

Several molecules that attach to special receptors called clathrin coated pits on the outside of cells cause the cell to perform endocytosis, an invagination of the plasma membrane to incorporate the molecule and associated structures into endosomes. This mechanism is used for three main purposes:

Uptake of essential

metabolites, for example, LDL. Uptake of some hormones and growth factors, for example, epidermal growth factor and nerve growth factor. Uptake of proteins that are to be destroyed, for example, antigens in phagocytotic cells like macrophages.

Receptor-mediated endocytosis can also be "abused":

Some

viruses, for example, the Semliki forest virus, enter the cell through this mechanism. Cholera, diphtheria, anthrax, tetanus, botulinum, and

other bacterial toxins enter the cell this way.

Protein destruction

Defective proteins are occasionally produced, or they may be damaged later, for example, by oxidative stress. Damaged proteins can be recycled. Proteins can have very different half lifes, mainly depending on their N-terminal amino acid residue. The recycling mechanism is mediated by ubiquitin.

Protein targeting in bacteria

With some exceptions, Bacteria lack membrane-bound organelles as found in eukaryotes, but they may assemble proteins onto various types of inclusions such as gas vesicles and storage granules. Bacteria may have a single plasma membrane (Gram-positive bacteria), or an inner membrane plus an outer membrane separated by the periplasm (Gram-negative bacteria). Proteins may be incorporated into the plasma membrane, or either trapped in the periplasm or secreted into the environment, according to whether or not there is an outer membrane. The basic mechanism at the plasma membrane is similar to the eukaryotic one. In addition, bacteria may target proteins into or across the outer membrane. Systems for secreting proteins across the bacterial outer membrane may be quite complex and play key roles in pathogenesis. These systems may be described as type I secretion, type II secretion, etc.

In most Gram-positive bacteria, certain proteins are targeted for export across the plasma membrane and subsequent covalent attachment to the bacterial cell wall. A specialized enzyme, sortase, cleaves the target protein at a characteristic recognition site near the protein C-terminus, such as an LPXTG motif (where X can be any amino acid), then transfers the protein onto the cell wall. An system analogous to sortase/LPXTG, termed exosortase/PEP-CTERM, is proposed to exist in a broad range of Gram-negative bacteria.

Secretory pathways

The secretory pathway includes vesicular traffic, secretion, and endocytosis. Secretory proteins follow this pathway.

Early stages

Retrograde transport is common in the early stages. Proteins that have been successfully delivered to the Golgi apparatus advance through cisternal progression.

Later stages

Coated vesicles mediate several transport steps.

References

1. Kanner EM, Friedlander M, Simon SM. (2003). "Co-translational targeting and translocation of the amino terminus of opsin across the endoplasmic membrane requires GTP but not ATP". J. Biol. Chem. 278 (10): 7920–7926.

doi:10.1074/jbc.M207462200. PMID 12486130. 2. Kanner EM, Klein IK. et al. (2002). "The amino terminus of opsin translocates "posttranslationally" as efficiently as cotranslationally". Biochemistry 41 (24): 7707–7715. doi:10.1021/bi0256882. PMID 12056902.

(From Wikipedia, the free encyclopedia)

Protein targeting (1)

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This article deals with protein targeting in eukaryotes except where noted.

Protein targeting or protein sorting is the mechanism by which a cell transports proteins to the appropriate positions in the cell or outside of it. Sorting targets can be the inner space of an organelle, any of several interior membranes, the cell's outer membrane, or its exterior via secretion. This delivery process is carried out based on information contained in the protein itself. Correct sorting is crucial for the cell; errors can lead to diseases.

Targeting signals

Targeting signals are the pieces of information that enable the cellular transport machinery to correctly position a protein inside or outside the cell. This information is contained in the polypeptide chain or in the folded protein. The continuous stretch of amino acid residues in the chain that enables targeting are called signal peptides or targeting peptides. There are two types of targeting peptides, the presequences and the internal targeting peptides. The presequences of the targeting peptide are often found at the N-terminal extension and is composed of between 6-136 basic and hydrophobic amino acids.In case of peroxisomes the targeting sequence is on the C-terminal extension mostly. Other signals are composed by parts which are separate in the primary sequence. To function, these components have to come together on the protein surface by folding. They are called signal patches. In addition, protein modifications like glycosylations can induce targeting.

Protein translocation

In 1970, Günter Blobel conducted experiments on the translocation of proteins across membranes. He was awarded the 1999 Nobel prize for his findings. He discovered that many proteins have a signal sequence, that is, a short amino acid sequence at one end that functions like a postal code for the target organelle. The translation of mRNA into protein by a ribosome takes place within the cytosol. If the synthesized proteins "belong" in a different organelle, they can be transported there in either of two ways, depending on the protein.

Cotranslational translocation

The N-terminal signal sequence of the protein is recognized by a signal recognition particle (SRP) while the protein is still being synthesized on the ribosome. The synthesis pauses while the ribosome-protein complex is transferred to a SRP receptor on the endoplasmic reticulum (ER), a membrane-enclosed organelle. There, the nascent protein is inserted into the Sec61 translocation complex (also known as the translocon) that passes through the ER membrane. The signal sequence is immediately cleaved from the polypeptide once it has been translocated into the ER by signal peptidase in secretory proteins. This signal sequence processing differs for some ER transmembrane proteins. Within the ER, the protein is first covered by a chaperone protein to protect it from the high concentration of other proteins in the ER, giving it time to fold correctly. Once folded, the protein is modified as needed (for example, by glycosylation), then transported to the Golgi apparatus for further processing and goes to its target organelles or is retained in the ER by various ER retention mechanisms.

Posttranslational translocation

Even though most proteins are cotranslationally translocated, some are translated in the cytosol and later transported to their destination. This occurs for proteins that go to a mitochondrion, a chloroplast, or aperoxisome (proteins that go to the latter have their signal sequence at the C terminus). Also, proteins targeted for the nucleus are translocated post-translation. They pass through the nuclear envelope via nuclear pores.

Transmembrane proteins

The amino acid chain of transmembrane proteins, which often are transmembrane receptors, passes through a membrane one or several times. They are inserted into the membrane by translocation, until the process is interrupted by a stop-transfer sequence, also called a membrane anchor sequence. These complex membrane proteins are at the moment mostly understood using the same model of targeting that has been developed for secretory proteins. However, many complex multi-transmembrane proteins contain structural aspects that do not fit the model. Seven transmembrane G-protein coupled receptors (which represent about 5% of the genome of humans) mostly do not have an amino-terminal signal sequence. In contrast to secretory proteins, the first transmembrane domain acts as the first signal sequence, which targets them to the ER membrane. This also results in the translocation of the amino terminus of the protein into the ER membrane lumen. This would seem to break the rule of "co-translational" translocation which has always held for mammalian proteins targeted to the ER. This has been demonstrated with opsin with in vitro experiments. A great deal of the mechanics of transmembrane topology and folding remains to be elucidated.

Sorting of proteins to mitochondria

Most mitochondrial proteins are synthesized as cytosolic precursors containing uptake peptide signals. Cytosolic chaperones deliver preproteins to channel linked receptors in the mitochondrial membrane. Thepreprotein with presequence targeted for the mitochondria is bound by receptors and the General Import Pore (GIP) (Receptors and GIP are collectively known as Translocase of Outer Membrane or TOM) at theouter membrane. The preprotein is translocated through TOM as hairpin loops. The preprotein is transported through the intermembrane space by small TIMs (which also acts as molecular chaperones) to the TIM23 or 22 (Translocase of Inner Membr

ane) at the inner membrane. Within the matrix the targeting sequence is cleaved off by mtHsp70. Three mitochondrial outer membrane receptors are known:

TOM20, TOM22 and TOM70
TOM70: Binds to internal targeting peptides and acts as a docking point for cytosolic chaperones.

TOM20: Binds presequences
TOM22: Binds both presequences and internal targeting peptides

The TOM channel is a cation specific high conductance channel with a molecular weight of 410 kDa and a pore diameter of 21Å.

The presequence translocase23 (TIM23) is localized to the mitochondial inner membrane and acts a pore forming protein which binds precursor proteins with its N-terminal. TIM23 acts a translocator for preproteins for the mitochondrial matrix, the inner mitochondrial membrane as well as for the intermembrane space. TIM50 is bound to TIM23 at the inner mitochondrial side and found to bind presequences. TIM44 is bound on the matrix side and found binding to mtHsp70.

The presequence translocase22 (TIM22) binds preproteins exclusively bound for the inner mitochondrial membrane.

Mitochondrial matrix targeting sequences are rich in positively charged amino acids and hydroxylated ones.

Proteins are targeted to submitochondrial compartments by multiple signals and several pathways.

Targeting to the outer membrane, intermembrane space, and inner membrane often requires another signal sequence in addition to the matrix targeting sequence.

Sorting of proteins to chloroplasts

The preprotein for chloroplasts may contain a stromal import sequence or a stromal and thylakoid targeting sequence. The majority of preproteins are translocated through the Toc and Tic complexes located within the chloroplast envelope. In the stroma the stromal import sequence is cleaved off and folding as well as intra-chloroplast sorting to thylakoids continues. Proteins targeted to the envelope of chloroplasts usually lack cleavable sorting sequence.

Selection and amplification binding assay

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Selection and amplification binding assay (SAAB) is a molecular biology technique developed by T. Keith Blackwell and Harold Weintraub in 1990. Its major use is to find the DNA binding site for proteins.

Experimental details

SAAB experimental procedure consists of several steps, depending upon the knowledge available about the binding site. A typical SAAB consists of the following steps

I. Synthesis of template with random sequence in binding site Three situations are possible: (i) when both the binding site and the protein are known and available; (ii) when only a consensus binding site is available and the binding protein is not; and (iii) when the protein is available, but the binding site is unknown. When the binding site is not known, the number of random nucleotide positions in the template must be large

II. Incubate labeled double stranded template with protein Usually the protein has to synthesis in a host cell with fusion techniques. Longer incubation time and large quantity are provided in case of unspecific binding site.

III. Isolate the DNA bound protein by EMSA The DNA bound protein , migrated in aralamide gel is isolated by autoradiography as per Electrophoretic mobility shift assay (EMSA) protocol.

IV. Amplify the bound template by PCR. For positive control, amplify the starting template also The bound DNA is isolated via gel excision and purified, and amplified using PCR.

V. Label amplified binding site and reselect for binding by EMSA (Usually 5 times)

Precede the binding step at least for 5 times with the amplified labeled DNA sample and fusion protein.

VI. Sequence the DNA After final step of selection and electrophoresis, clone the DNA into some cloning vector and sequence it. Originally Blackwell used Pyro sequencing, which can be replaced by modern techniques.

Identification of Quox_1 Homeodomain DNA Binding Sequence Using SAAB (A descriptive example from resent study)-An example Quox1 is a novel homeobox gene (A homeobox is a DNA sequence found within genes that are involved in the regulation of patterns of development (morphogenesis) in animals, fungi and plants, originally isolated from cDNA library of five week quail (Quail is a collective name for several genus of mid-sized birds) embryo. It is the only gene in the hox family that has been found to express in both prosencephalon and mesencephalon involved in the differentiation of the central and peripheral nerve cells. The optimal DNA binding site for Quox1 or its mammalian homologs was identified by SAAB in 2004. Quox1 homeobox sequence was obtained by PCR amplification from a human embryo cDNA library by standard procedure. The amplified DNA fragment was digested with EcoRV and XhoI and cloned into the SmaI and XhoI restriction site of the expression vector pGEMEXxBal. The recombinant plasmids were transformed into competent Escherichia coli strain BL21 and Quox1 fusion proteins were isolated by chromatographic techniques.

The radio labeled probe was incubated with 25 pmol of purified Quox1 homeodomain fusion protein in binding buffer for EMSA. The protein bound DNA was detected by autoradiography, and the bands representing protein–DNA complexes were excised from the gel and the eluted DNA were amplified by PCR using primers complementary to the 20 bp nonrandom flanking sequences. After 5 set of the same procedure,the purified DNA was cloned into pMD 18T and sequenced. Finally the sequence CAATC was identified as the consensus binding sequence for Quox1 homeodomain.

Applications of the SAAB

By combining the power of random-sequence selection with pooled sequencing, the SAAB imprint assay makes possible simultaneous screening of a large number of binding site mutants. SAAB also allows the identification of sites with high relative binding affinity since the competition is inherent in the protocol. It can also identify site positions that are neutral or specific bases that can interfere with binding, such as a T at - 4 in the E47 half-site. We can apply the technique to less affinity binding sequence also, provided to keep high concentration of binding protein at each step of binding. It is also possible to identify the binding site even if both the protein and sequence is not known.

References

^ Blackwell, K. T., and Weintraub, H. (1990) Science, 250,1104–1110

^ Amendt, B.A., L.B. Sutherland, and A.F. Russo, Transcriptional Antagonism between Hmx1 and Nkx2.5 for a Shared DNA-binding Site. Journal of Biological Chemistry, 1999. 274(17): p. 11635-11642.

^ Rienhoff, H.Y., Identification of a transcriptional enhancer in a mouse amyloid gene. Journal of Biological Chemistry, 1989. 264(1): p. 419-425.

^ Kim, T.-G., et al., JUMONJI, a Critical Factor for Cardiac Development, Functions as a Transcriptional Repressor. Journal of Biological Chemistry, 2003. 278(43): p. 42247-42255.

Reverse transfection

2011-06-30T21:49:00.000-07:00

The term reverse transfection comes from the invention and development of a microarray-driven gene expression system by Junald Ziauddin and David M. Sabatini in 2001. As DNA are printed on a glass slide for transfection process to occur before the addition of adherent cells, the order of addition of DNA and adherent cells is a reverse of that of conventional transfection. Hence the word “reverse” is used.

Reverse Transfection Process

Preparation of transfection mix for printing onto a slide

DNA-gelatin mixture can be used for printing onto a slide: Gelatin powder is first dissolved in sterile MilliQ water to form 0.2% gelatin solution. Purified DNA plasmid is then mixed with gelatin solution and the final gelatin concentration is kept greater than 0.17%. Besides the use of gelatin, atelocollagen and fibronetin are also successful transfection vectors for introducing foreign DNA into the cell nucleus.

Printing of DNA-gelatin mixture onto a slide

After the DNA-gelatin mixture preparation, the mixture is pipetted onto a slide surface and then the slide is put into a petri dish with a cover. A drying chemical is added into the dish to dry up the solution. Finally, cultured cells are poured into the dish for plasmid uptake. However, with the invention of different types of microarray printing system, hundreds of transfection mixes containing different DNA of interest can be printed on the same slide for the uptake of plasmids by cells. There are 2 major kinds of microarray printing systems manufactured by different companies: Contact and non-contact printing system.

An example of non-contact printing system is Piezorray Flexible Non-contact Microarraying System. It uses pressure control and a piezoelectric collar to squeeze out consistent drops of approximately 333 pL volume. The PiezoTip dispensers do not contact the surface to which the sample is dispensed, thus contamination potential is reduced and the risk of disrupting the target surface is eliminated. An examples of contact printing system is SpotArray 72 (Perkin Elmer Life Sciences, Inc) contact spotting system. Its printhead can accommodate up to 48 pins and creates compact arrays by selectively raising and lowering subsets of pins during printing. After printing, the pins are washed with a powerful pressure-jet pin washer and then vacuum-dried, eliminating carryover. Another example of contact printing system is Qarray system (Genetix Inc). It has three types of printing systems, QArray Mini, QArray 2 and QArray Max.

After printing, the solution is allowed to dry up and the DNA-gelatin is sticked tightly at the position on the array.

Use of HybridWell for reverse transfection of gelatin-DNA into cells on a slide

Firstly, the adhesive from the HybriWell is peeled off and then attach the HybriWell over the area of the slide printed with the gelatin-DNA solution. Secondly, pipette 200ul of transfection mix into one of the ports of HybriWell. The mix will distribute evenly over the array. Incubate the array at particular temperature and time length depending on the types of cells used. Thirdly, pipette away the transfection mix and pull off the HybriWell using a thin tipped forceps. Fourthly, put the printed slide treated with transfection reagent into a square dish with the printed-side facing up. Fifthly, gently pour the harvested cells onto the slides (don’t pour directly on the printed areas). Finally, place the dish in a 37oC, 5% CO2 humidified incubator and incubate overnight.

Other transfection reagents for reverse transfection

Effectene Reagent is used in conjunction with the Enhancer and the DNA condensation buffer (Buffer EC) to achieve high transfection efficiencies. In the first step of Effectene–DNA complex formation, the DNA is condensed by interaction with the Enhancer in a defined buffer system. Effectene Reagent is then added to the condensed DNA to produce condensed Effectene–DNA complexes. The Effectene–DNA complexes are mixed with medium and directly added to the cells. Effectene Reagent spontaneously forms micelle structures that show no size or batch variation, as found with preformulated liposome reagents. This unique feature ensures excellent reproducibility of transfection complex formation. The process of highly condensing DNA molecules and then coating them with Effectene Reagent is a particularly effective way to transfer DNA into eukaryotic cells.

Advantages and disadvantages of reverse transfection techniques

The advantages of reverse transfection than conventional transfection are: Firstly, the addition and attachment of target cells to the DNA-loaded surface can lead to higher probability of cell-DNA contact, potentially leading to higher transfection efficiencies. Secondly, less labour-intensive and saving materials. Less DNA is required for the success of transfection. Thirdly, high throughput screening: each time hundreds of genes can be expressed in cells together on a single microarray for studying gene expression and regulation. Fourthly, parallel cell seeding in a single chamber for 384 experiments together with no physical separation between experiments increases the screening data quality. Well-to-well variations occur in experiments done in multiwall dishes. Fifthly, exact replicate arrays can be produced as same sample source plate can be dried and printed on different slides for storage at least 15months without apparent loss of transfection efficiency.

There is a disadvantage of using reverse transfection: Reverse transfection is more expensive because highly accurate and efficient microarray printing system is needed to print the DNA-gelatin solution onto the slides. Firstly, applications with different cell lines have so far required variations in the protocols to manufacture siRNA or plasmid arrays, which involves a considerable amount of development and testing. Secondly, possibility of cross-contamination of the array spots when spot densities increase; therefore, optimization of the array layout is important.

shRNA transfection (2)

2011-06-30T21:48:00.001-07:00

Transfection Reagents

§ Cationic lipid

Cationic lipid transfection reagents are suitable for transfecting into a wide variety of dividing cell cultures. Commercial examples include: Lipofectamine / L2000, Dharmafect, iFect, and TransIT TKO. Cationic lipids work by forming lipsomal vesicles that house the siRNA payload and bleb their way through the living cell membrane and into the cytoplasm. The efficiency of this process must be determined in order to have confidence in the knockdown effects. There are numerous commercial sources for transfection reagents for good reason; there are numerous cell types and lipsome structure will influence transfection efficiency in the multitude of experimental cell types that exist.

§ Polymeric

Polymeric formulations have been developed and optimized for transfection of shRNA plasmid DNA into the nucleus of cultured eukaryotic cells by vendors such as Open Biosystems. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus (Pollard et al 1998). Polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.

shRNA Vector Transfection

§ In a six well tissue culture plate, grow cells to a 50-70% confluency in antibiotic-free normal growth medium supplemented with FBS.

NOTE: This protocol is recommended for a well from a 6 well tissue culture plate. Adjust cell and reagent amounts proportionately for wells or dishes of different sizes.

NOTE: Healthy and subconfluent cells are required for successful transfection experiments. It is recommended to ensure cell viability one day prior to transfection.

Prepare the following solutions:

NOTE: The optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio should be determined experimentally beginning with 1 μg of shRNA Plasmid DNA and between 1.0 and 6.0 μl of shRNA Plasmid Transfection Reagent as outlined below. Once the optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio has been identified for a given cell type, the appropriate amount of shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent complex used per well should be tested to determine which amount provides the highest level of transfection efficiency. For example, if the optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio is 1 μg:1 μl, then amounts ranging from 0.5 μg/0.5 μl to 2.0 μg/2.0 μl should be tested.

Solution A: For each transfection, dilute 10 μl of resuspended shRNA Plasmid DNA (i.e. 1 μg shRNA Plasmid DNA) into 90 μl shRNA Plasmid Transfection Medium (serum antibiotic free medium).

Solution B: For each transfection, dilute 1 - 6 μl of shRNA Plasmid Transfection Reagent with enough shRNA Plasmid Transfection Medium to bring final volume to 100 μl.

NOTE: Do not add antibiotics to the shRNA Plasmid Transfection Medium.

NOTE: Optimal results may be achieved by using siliconized microcentrifuge tubes.

NOTE: Although highly efficient in a variety of cell lines, not all shRNA Plasmid Transfection Reagents may be suitable for use with all cell lines.

§ Add the shRNA Plasmid DNA solution (Solution A) directly to the dilute shRNA Plasmid Transfection Reagent (Solution B) using a pipette. Mix gently by pipetting the solution up and down and incubate the mixture 15-45 minutes at room temperature.

§ Wash the cells twice with 2 ml of shRNA Transfection Medium. Aspirate the medium and proceed immediately to the next step. NOTE: Do not use PBS as the residual phosphate may compete with DNA and bind the shRNA Plasmid Transfection Reagent, thereby reducing the transfection efficiency.

NOTE: Do not use PBS as the residual phosphate may compete with DNA and bind the shRNA Plasmid Transfection Reagent, thereby reducing the transfection efficiency. For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well.

§ For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well.

§ Add the 200 μl shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent Complex (Solution A + Solution B) dropwise to well, covering the entire layer.

§ Gently mix by swirling the plate to ensure that the entire cell layer is immersed in solution.

§ Incubate the cells 5-7 hours at 37° C in a CO2 incubator or under conditions normally used to culture the cells.

NOTE: Longer transfection times may be desirable depending on the cell line.

§ Following incubation, add 1 ml of normal growth medium containing 2 times the normal serum and antibiotics concentration (2x normal growth medium).

§ Incubate the cells for an additional 18-24 hours under conditions normally used to culture the cells.

Aspirate the medium and replace with fresh 1x normal growth medium.

§ Assay the cells using the appropriate protocol 24-72 hours after the addition of fresh medium in the step above.

NOTE: Controls should always be included in shRNA experiments. Control shRNAs are available as 20 μg. Each encode a scrambled shRNA sequence that will not lead to the specific degradation of any known cellular mRNA.

NOTE: For Western blot analysis prepare cell lysate as follows: Wash cells once with PBS. Lyse cells in 300 μl 1x Electrophoresis Sample Buffer (sc-24945) by gently rocking the 6 well plate or by pipetting up and down. Sonicate the lysate on ice if necessary.

NOTE: For RT-PCR analysis isolate RNA using the method described by P. Chomczynski and N. Sacchi (1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159) or a commercially available RNA isolation kit.

Lentivrus Infection

Materials:

§ Target cells are SH-SY5Y human neuroblastoma cells.

§ Complete medium: DMEM F-12 medium with 10% serum and Penicillin/Streptomycicn

§ Vectors are copGFP control Lentiviral Particles (sc-108084)

§ Use 24 well plate. Each well has 1.9 mm2, good for working with 0.5 ml medium.

Concentration Considerations:

§ Test 5μg/ml Polybrene

§ Each well contains 4×104 cells. Will use Viral Particles at a quantity of 4×105 infection units. The concentration of provided viral particles was 5000 infections units per μl, so I will try 100 μl. The MOI is 10.

Day 1 Seed Cells

§ Plate target cells in 12 well plate 24 hours prior to viral infection. Each well contains 4×104 cells in 0.5 ml complete medium.

Day 2 Lentiviral Infection

§ Monitor the seeded cells and make sure that the cells are around 50% confluent.

§ Bring Polybrene (sc-134220) to room temperature, and complete medium to 37°C.

§ Prepare a mixture of complete medium with Polybrene at a final concentration of 5 μg/ml.

§ Remove media from plate wells and replace with 1ml of Polybrene/media mixture per well.

§ Put the plate back to the incubator until lentiviral particles were thawed.

§ Thaw lentiviral particles at room temperature and mix gently before use. This takes about 5 minutes.

§ Infect cells by adding the indicated amount of shRNA lentiviral particles to the culture.

§ Swirl the plate gently to mix and incubate overnight.

NOTE: Lentiviral particles were thawed at room temperature. As soon as the vial is thawed, Lentiviral particles were immediately added to the plate to avoid prolonged exposure of the particles to ambient temperature. Did not use ice.

Day 3 Change Medium

§ Remove the culture medium and replace with 1 ml of complete medium (without Polybrene).

§ Incubate the cells for 2 days.

§ Examine GFP positive cells under microscope. Found that around 80% cells were GFP-positive, the signal was weak.

Day 5 and on Culturing of Cells before Selection

§ Starting from Day 5, change to fresh medium and wait for cells to reach confluency in order to get enough cells for the selection.

Changed medium on day5, day8.

shRNA Controls

Negative Controls

§ Untreated Cells. Untreated cells will provide a reference point for comparing all other samples.

§ Empty construct, containing no shRNA insert; The empty viral particles or DNA are a useful negative control that will not activate the RNAi pathway because it does not contain an shRNA insert. It will allow for observation of cellular effects of the transduction/transfection process. Cells transduced/transfected with the empty control provide a useful reference point for comparing specific knockdown.

§ Non-targeting shRNA; This non-targeting shRNA is a useful negative control that will activate RISC and the RNAi pathway, but does not target any human or mouse genes. The short hairpin sequence cotnains 5 base pair mismatches to any known human or mouse gene. This allows for examination of the effects of shRNA transduction/transfection on gene expression. Cells transduced/transfected with the non-target shRNA will also provide useful reference for interpretation of knockdown.

Positive Controls

§ Positive shRNA knockdown control; This control contains shRNA sequence that targets GFP expression. This shRNA control has been experimentally shown to reduce GFP expression. This control serves to quickly visualize knockdown in cells expressing GFP.

§ Positive shRNA knockdown control; This control contains shRNA sequence that targets eGFP expression (GenBank Accession # pEGFP U476561). The shRNA has been experimentally shown to reduce eGFP expression by 90% in C166-GFP mouse fibroblast cells 48 hours post-transduction by mRNA transcript level. This control serves to quickly visualize knockdown in cells expressing eGFP.

§ Positive reporter vector or lentiviral particles; This is a useful positive control for measuring transduction/transfection efficiency and optimizing shRNA delivery. The GFP Control contains a gene encoding GFP, driven by the CMV promoter. This control provides fast visual confirmation of successful transduction/transfection.

copGFP

The copGFP protein is a novel natural green monomeric green fluorescent protein cloned from copepod Pontellina plumata, a type of plankton. The copGFP protein is a non-toxic, non-aggregating protein with fast protein maturation, high stability at a wide range of pH (pH 4-12), and fluorescent properties that do not require any additional cofactors or substrates.

Due to its exceptional properties, copGFP is an excellent fluorescent marker that can be used instead of EGFP (the widely used Aequrea victoria GFP mutant) for monitoring delivery of lentiviral constructs into cells. The copGFP protein has a very bright fluorescence that exceeds the brightness of EGFP by approximately a third.

The copGFP protein emits green fluorescence with the following characteristics:

§ emmision wavelength max – 502 nm

§ excitation wavelength max – 482 nm

§ quantum yield – 0.6

§ extinction coefficient – 70,000 M-1 cm-1

When assaying cells, DO NOT fix with methanol and minimize exposure to light. PFA/Formalin fixation works.

Factors Influencing Successful Transfection

Concentration and purity of nucleic acids

Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids.

DNA:In terms of plasmid preparation, McManus Lab has not observed a need to use E. coli cells that are highly defective for recombination. High DNA quality usually means high transfection efficiency. All DNA preparations should be performed by Cesium prep or endotoxin-free ion exchange plasmid purification methods. If poor transfection is consistently observed, it may be worth performing a additional clean-up of the DNA. The transfection protocols described here are sensitive to the amount of DNA. It is important to optimize DNA:Transfection Reagent ratios.

Transfection in serum-free media

The highest transfection efficiencies can be obtained if the cells are exposed to the transfection complexes in serum free conditions followed by the addition of medium containing twice the amount of normal serum to the complex medium 3–5 hrs post transfection (leaving the complexes on the cells). However, the transfection medium can be replaced with normal growth medium if high toxicity is observed.

No antibiotics in transfection medium

The presence of antibiotics can adversely affect the transfection efficiency and lead to increased toxicity levels in some cell types. It is recommended that these additives be initially excluded until optimized conditions are achieved, then these components can be added, and the cells can be monitored for any changes in the transfection results.

High protein expression levels

Some proteins when expressed at high levels can by cytotoxic; this effect can also be cell line specific.

Cell history, density, and passage number

It is very important to use healthy cells that are regularly passaged and in growth phase. The highest transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.

shRNA transfection (1)

2011-06-30T21:47:00.000-07:00

shRNA Transfection

Lentivirus, shRNA plasmid, or siRNA?

Choosing between Lentivirus, shRNA transfer vector, or siRNA, depends on what you are seeking to accomplish. What advantage is there for you to create a stable knockdown versus a transient knockdown? If you are running your RNAi on an easy to transfect cell line, under a transient RNAi event, then siRNA is a well defined, proven and effective method. If you are working with a primary cell, neuron, or generally hard to transfect cell, then lentiviral particles for introducing shRNA is attractive. The shRNA transfer vector alone in theory should be easy to work with insofar as culturing the cell, DNA transfection, antibiotic selection, and then collect data. However the true purpose of the lentivirus transfer vector is for packaging into lenti particles.

Establishing a stable knockdown phenotype is feasible with the use of lentiviral particles. Viral particles that have a VSV-G coat protein (Santa Cruz Biotechnology Inc.) will have broad tropism. Otherwise if you are studying effects that can be measured with transient knockdown, then siRNA is a more simple and straightforward approach. siRNA is a user friendly technique since you can calculate empirical moles of duplex/# cells or total volume (molarity), with a minimal number of steps and peripheral controls toward achieving results.

shRNA transfer vectors are ~7 kb DNA constructs that can give less stability problem since it is DNA instead of RNA. However there are considerable nuances in the actual design and cloning of these vectors, in the actual establishment of antibiotic resistance toward generating a stable cell, and in controlling for the specific silencing/reproducible results. shRNA has limitations due to the nature of having such a large delivery vector producing a small hairpin substrate, and over cell passages under antibiotic selective pressure (ie puro resistance may not = Pol III shRNA cassette expression).

TECHNICAL SERVICE GUIDE: siRNA

Catalog # Lot #

Summary:

1) Optimize the transfection reagent; measure transfection efficiency of the transfection reagent with FITC-siRNA.

2) Measure knockdown in a range of cell densities ( 30-80%) within 24-72 hours

3) Measure knockdown in a range of siRNA concentrations (30-90 nM) within 24-72 hours

Providing suggestions outlined in the notes below is worth considering and may bring success.

Background Info

§ What are the experimental results?

§ Describe how gene knockdown is measured? qPCR / Western / IF

§ How was the RNA reconstituted?

NOTE: siRNA ships lyophilized along with RNase free water with instructions to reconstitute with 330 ul of H2O to make 10 uM solution. Having the correct molarity of the solution is critical.

§ Molarity of siRNA vialed: 10 uM ( uM/L )

§ Volume after reconstitution: 330 uL

§ Mass of 1 mole of siRNA: 13800 g/mol ( 21nt X 660 g/base pair)

§ Total mols per vial: 10 um/L X 330 uL = 3.3 nm

§ Total grams per vial: 3.3 nm X 13800 g/mol = 45.5 ug

§ Solution concentration: 45.5 ug/ 330 uL = 0.138 ug/uL

§ Did this same vial or other lot of siRNA work in the past?

NOTE: If the siRNA same cat# has worked in the past, and now does not work, this may suggest RNase contamination. There are ways to determine this by running 1 pmol (17 ng) siRNA in a native 2% agarose gel, however replacing the vial is a straightforward solution.

Transfection Efficiency

§ Describe the cell type for this experiment?

§ What transfection reagent is used for the siRNA tranfection?

NOTE: Cationic lipid based transfection reagents (ie Lipofectimine, L2000, Transit TKO, Oligofect, Dharmafect, sc-29528) are each one a unique formula. Certain cell types will respond better to certain cationic lipid (positive charge lipophilic) reagents. For this reason, measuring transfection efficiency is necessary.

§ How was transfection efficiency measured?

NOTE: The researcher may have an existing transfection reagent that works on their cells in other experiments (ie cDNA). Suggest to try the same reagent and measure transfection efficiency.

§ What time point was transfection uptake of FITC-siRNA measured?

NOTE: Measuring transfection efficiency with sc-36869 will validate that liposome-dependent siRNA entry into the cells is taking place efficiently. It is important to measure transfection efficiency 5-7 hours post transfection since this is when the optimum time point where most transfection takes place. Common methods are IF or Flow cytomtetry.

Cell Confluency

§ Adherent cell (grows on the surface of the plate): What is the cell confluency at time of transfection?

§ Suspension cell (ie leukocytes/lymphocytes, cells are suspended in the media) : How many cell count # used to seed the well?

NOTE: A hemocytometer (cell counter) is common for counting cells for seeding into multiwell plates (6, 12, 24 well); originally designed for performing blood cell counts. Cell density is an important parameter for knockdown. Optimum cell density will vary and typically falls between 30-80%. NOTE: Setting up a 6 or 12 well experiment and trying a range of cell confluencies (30, 50, 70%), will reveal an optimal cell density where knockdown is optimal with minimal cell death. Effective confluence can range from 30-80%.

siRNA Concentration

§ What nanomolar concentration(s) of siRNA are tested?

NOTE: Setting up a 6 or 12 well experiment and trying a range of cell confluencies (30, 50, 70%) & a range of siRNA concentrations (30, 60, 90 nM) will reveal an optimal convergence of cell density and concentration of siRNA where knockdown is optimal with minimal cytotoxicity (cell death).

§ What time points is RNAi measured?

NOTE: 48 hours post transfection is a relevant singular point. Measuring knockdown for a few time points in the 24-72 hour window may indicate the frame when RNAi is most optimal. Titrating the siRNA concentration (30-90 nM) for the cells will indicate the best amount to see an effect.

Measuring Knockdown

§ How is RNAi measured? Western blot - IF - qPCR - other

NOTE: For WB, titrating the antibody may reveal subtle changes in knockdown. For IF, running secondary controls may indicate nonspecific fluorescence mistaken for signal.

§ Quantitative RT-PCR, which primers were used and what type of system?

NOTE: With appropriate internal controls (GAPDH, DNA contamination control), qPCR can be very reliable in determining translation initiation arrest.

TECHNICAL SERVICE GUIDE: Lentivirus

Catalog # Lot #

Summary:

1) Determine if the VSV-G coat protein has tropism toward the target cell; measuring transduction efficiency with sc-108084 2) Measure a range of MOI (5-10+) 3) Measure knockdown within 48-72 hours after puromycin selection

Measure transduction efficiency

§ Transduction in what cell type?

§ Primary cell or Continuous/immortal cell?

NOTE: Primary cell cultures are first generation cells from a living organism and typically have less than 5 passage lifespan. Lenti is popular for primary cells since they are difficult to transfect. Continuous or immortalized cells have the ability to proliferate indefinitely in culture.

§ Is this cell type known to have tropism for VSV-G coat protein?

§ How is transduction efficiency measured for tropism to VSV-G?

§ If a copGFP expressing Lentivirus was used to measure tropism, at what time point was transduction efficiency of the copGFP Control Lentiviral Particles or other reporter measured?

NOTE: 48 hours post-transduction is the time point where puromycin selection begins. This is also a good time point to evaluate transduction efficiency by measuring copGFP fluorescence inside the cells.

§ How was the reporter gene measured? (FCM, IF, other)

Multiplicity of Infection (MOI)

X = How many cell count was transduced? NOTE: A hemocytometer is common for this step; originally designed for performing blood cell counts, contains a etched grid on a slide, count cells/square in 5-10 squares, then average out the number and extrapolate. Y = How many uL of virus was used? NOTE: MOI = X / (Y * (particles/uL)). HEK293T and other easy to transduce cells (MOI of 5-20), while neuronal cells,SHSY5Y, may require MOI of 10-50.

Puromycin Selection

§ How many [ug/ml] puromycin is added at what time point post transduction?

§ How was optimum puromycin concentration determined?

NOTE: The minimum antibiotic concentration to use is the lowest concentration that kills 100% of non-transfected cells in 3-5 days from the start of puromycin selection (normal range; 1-10 ug/ml). Add puromycin 48 hours post transduction.

§ Western blot, IF or Quantitative RT-PCR?

§ Negative controls (scrambled hairpin virus, no virus)?

NOTE: Running a parallel transduction with no virus should yield 100% cytotoxicity upon puromycin addition. Scrambled hairpin virus (sc-108080) transduction is useful to determine if any other aspect of the transduction process influences knockdown, including the presence of a non gene specific hairpin (nonspecific antisense).

Transient shRNA Transfection

Instead of chemically synthesizing the siRNAs before introducing it in the cell, the siRNAs are made directly by the cells through an expression vector that is transiently transfected into a dividng cell. The shRNA transfer vector alone can be transiently introduced into the dividing cell where the shRNA is synthesized by cellular machinery. While transient transfection is advantageous for fast analysis of shRNA mediated effects, stable transfection ensures long-term, reproducible as well as defined shRNA effects.

Stable shRNA Transfection

For many disease models, the most desirable cell types such as immune system or primary cells are not amenable to transfection. Viral delivery of RNAi vectors is a powerful alternative to transfection for these cell types as well as for in vivo applications. Stable expression is achieved by integration of the gene of interest into the target cell's chromosome: Initially the shRNA of interest has to be introduced into the cell, subsequently into the nucleus, and finally it has to be integrated into chromosomal DNA.

Stable expression can be influenced by two factors: The transfection method used and the vector containing the shRNA of interest. The transfection method determines which cell type can be targeted for stable integration through antibiotic selection. While many lipofection reagents transfect DNA up to a certain amount into adherent cell lines, efficient delivery of DNA into difficult-to-transfect suspension cell lines or even primary cells is only possible with viral methods and nucleofection.

Nucleofection

Nucleofection is a non-viral method of introducing DNA molecules into the nucleus of dividing cells, therefore significantly increasing the chances of chromosomal integration of the transgene. The technology is pioneered by Amaxa

Santa Cruz Biotechnology, Inc. does not disclose vector map information for the sh plasmids, including RE. This removes variable of a single cut RE in the empty showing up in a cloned-in sh,

The tech writing for Lonza nucleofection would suggest a linear plasmid has more efficient outcome for GFP expression , however

§ Values are so close between linear and circular DNA for the 2 and 5ug event looking at GFP expression and there is no claim that linear is necessary.

§ n= 2 cell lines tested provides limited insight into the validity of the claim for such a device as this : http://www.lonzabio.com/technology.html

§ Transgene study only - there is no mention of RNAi or lenti/sh vector suitability to this tech in their nucleofection literature -

Vector dependent

Although there is still some debate as to the effectiveness of this approach, a regular shRNA transfer vector may be able to integrate into the genome of the target cell by antibiotic selection alone. The process may occur randomly by the cell's machinery itself, possibly via DNA repair and recombination enzymes. If this phenomenon does occur, integration into inactive heterochromatin may result in little or no shRNA expression, whereas integration into active euchromatin may allow for shRNA expression. However, random integration could also lead to silencing of the shRNA cassette. Several strategies have been developed to overcome the negative position effects of random integration: Site-specific, homologous and transposon-mediated integration strategies are used but require the expression of integration enzymes or additional sequences on the plasmid.

Lentiviral particle dependent

Lentiviral particles are highly efficient at infection and stable integration of the shRNA into a cell system. To obtain the lentiviral particle, the transfer vector that contains the shRNA cassette is already flanked by LTRs and the Psi-sequence of HIV. The LTRs are necessary to integrate the shRNA cassette into the genome of the target cell, just as the LTRs in HIV integrate the dsDNA copy of the virus into its host chromosome. The Psi-sequence acts as a signal sequence and is necessary for packaging RNA with the shRNA into pseudovirus particles. Viral proteins which make virus shells are provided in the packaging cell line (HEK 293T), but are not in context of the LTRs and Psi-sequences and so are not packaged into virions. Thus, virus particles are produced that are replication deficient. Lentiviral particles can infect both dividing and nondividing cells because their preintegration complex (virus “shell”) can get through the intact membrane of the nucleus of the target cell.

§ Lentiviral systems efficiently transduce both dividing and non-dividing cells

§ Study long-term gene knockdown with stable expression

§ Reproducibly transduce cell populations

§ Inducible or constitutive gene knockdown

Weil’s myelin stain

2011-06-30T21:46:00.002-07:00

Description:

Weil’s stain is a modification for paraffin sections of the Weigert-Pal-Kulschitsky technique. The underlying principle of these methods involves the reduction of chrome salt to chromium dioxide by myelin. The chromium subsequently acts as a mordant for the haematoxylin, intensifying the stain.

Procedure:

This procedure is generally conducted on sections from formalin-fixed, paraffin-embedded tissue that are cut between 8-15 µm. Spinal cord tissue is rich in myelinated axons and can be used as a positive control.

1. Dewax and hydrate sections to distilled water.

2. Put slides in freshly prepared Staining Solution at 56-60C for 30 minutes.

3. Wash slides well in water.

4. Partially differentiate in iron alum differentiating solution until myelin sheaths stand out blueish-black on a pale grey background, approximately 5 minutes. If you are unsure, check your sections under the microscope at1 minute intervals).

5. Wash slides in tap water for 10 minutes.

6. Complete differentiation in Weigert’s differentiator, 1 to 2 minutes. Control this differentiation step carefully checking under the microscope, until the myelin is an intense deep blue against a creamy or clear background.

7. Wash well in tap water.

8. Dehydrate through a series of graded ethanol baths, clear in xylene, and mount.

Results:

Myelin-containing structures will be stained black, red blood cells will be black, nuclei will be blue, and the background should be clear or yellow.

Solutions:

Haematoxylin Solution, working strength

l 10g Haematoxylin

l 100 ml absolute ethanol.

l Allow solution to “ripen” naturally for six (6) weeks, forming the basis of a stock solution. Just prior to staining, create a working strength solution by diluting the stock 1:4 with distilled water

l Iron Alum Solution

l 4% (w/v) aqueous ferric ammonium sulphate.

Staining Solution

Mix equal volumes of preheated (56-60C) working strength Haematoxylin and Iron Alum solutions just prior to use.

Weigert’s Differentiator, 200 ml

1. Borax (sodium tetraborate), 2g

2. Potassium ferricyanide, 2.5g

3. Distilled water, 200ml

NCBI: Training & Tutorials

2011-06-30T21:39:00.000-07:00

NCBI：National Center for Biotechnology Information

U.S. government-funded national resource for molecular biology information. Access to many public databases and other references, ...
www.ncbi.nlm.nih.gov/

DATABASES

NCBI C++ Toolkit Manual

A comprehensive manual on the NCBI C++ toolkit, including its design and development framework, a C++ library reference, software examples and demos, FAQs and release notes. The manual is searchable online and can be downloaded as a series of PDF documents.

NCBI Education Page

Provides links to tutorials and training materials, including PowerPoint slides and print handouts.

NCBI Glossary

Part of the NCBI Handbook, this glossary contains descriptions of NCBI tools and acronyms, bioinformatics terms and data representation formats.

NCBI Handbook

An extensive collection of articles about NCBI databases and software. Designed for a novice user, each article presents a general overview of the resource and its design, along with tips for searching and using available analysis tools. All articles can be searched online and downloaded in PDF format; the handbook can be accessed through the NCBI Bookshelf.

NCBI Help Manual

Accessed through the NCBI Bookshelf, the Help Manual contains documentation for many NCBI resources, including PubMed, PubMed Central, the Entrez system, Gene, SNP and LinkOut. All chapters can be downloaded in PDF format.

NCBI Website Search

A database of static NCBI web pages, documentation, and online tools. These pages include such content as specialized online sequence analysis tools, back issues of newsletters, legacy resource description pages, sample code, and other miscellaneous resources. Searching this database is equivalent to a site search tool for the whole NCBI web site. FTP site is not covered.

DOWNLOADS

FTP: NCBI Field Guide Manual

Downloadable material for NCBI's previously offered Field Guide training course.

FTP: NCBI Structure Course Materials

PowerPoint slides, handouts and exercises for the previously offered NCBI course "Exploring 3D Molecular Structures."

NCBI Data Specifications

Specifications for NCBI data in ASN.1 or DTD format are available on the Index of data_specs page. The "NCBI_data_conversion.html" links to the conversion tool.

National Library of Medicine (NLM) DTDs

A suite of tag sets for authoring and archiving journal articles as well as transferring journal articles from publishers to archives and between archives. There are four tag sets: Archiving and Interchange Tag Set - Created to enable an archive to capture as many of the structural and semantic components of existing printed and tagged journal material as conveniently as possible; Journal Publishing Tag Set - Optimized for archives that wish to regularize and control their content, not to accept the sequence and arrangement presented to them by any particular publisher; Article Authoring Tag Set - Designed for authoring new journal articles; NCBI Book Tag Set - Written specifically to describe volumes for the NCBI online libraries.

TOOLS

Amino Acid Explorer

This tool allows users to explore the characteristics of amino acids by comparing their structural and chemical properties, predicting protein sequence changes caused by mutations, viewing common substitutions, and browsing the functions of given residues in conserved domains.

BLAST Tutorials and Guides

This page links to a number of BLAST-related tutorials and guides, including a selection guide for BLAST algorithms, descriptions of BLAST output formats, explanations of the parameters for stand-alone BLAST, directions for setting up stand-alone BLAST on local machines and using the BLAST URL API.

Coffee Break

Part of the NCBI Bookshelf, Coffee Break combines reports on recent biomedical discoveries with use of NCBI tools. Each report incorporates interactive tutorials that show how NCBI bioinformatics tools are used as a part of the research process.

E-Bench

This interactive tool allows users to build E-utility URLs, either from a form or by hand, and then view their raw output. The tool provides a simple environment for testing E-utility URLs before including them in applications.

Ebot

A tool that allows users to construct an E-utility analysis pipeline using an online form, and then generates a Perl script to execute the pipeline.

NCBI News

NCBI's monthly newsletter that provides information on new and updated databases, and software services. The News often has feature articles that highlight and demonstrate services, features, tools, and interesting data with practical examples of their use.

PSSM Viewer

Allows users to display, sort, subset and download position-specific score matrices (PSSMs) either from CDD records or from Position Specific Iterated (PSI)-BLAST protein searches. The tool also can align a query protein to the PSSM and highlight positions of high conservation.

PubMed Tutorials

A collection of web and flash tutorials on PubMed searching and linking, saving searches in MyNCBI, using MeSH and other PubMed services.

Science Primer

A basic introduction to the science and technology that underlies many of the NCBI resources. A great starting place for students and the general public, the Science Primer provides a basis for understanding the NCBI web site and mission, and provides direct links to many NCBI databases and tools. Topics include genome mapping, molecular modeling, mutations, microarrays (gene expression), genetics, pharmacogenomics (personalized medicine) and phylogenetics (evolutionary relationships).

C. Elegans Genetic Protocols

2010-12-25T05:57:00.002-08:00

C. elegans EMS Mutagenesis http://cobweb.dartmouth.edu/cgi-bin/cgiwrap/~ambros/protocol.cgi?id=26

Knockout Protocol

Genomic DNA prep

Integrating extrachromasomal arrays-- http://cobweb.dartmouth.edu/cgi-bin/cgiwrap/~ambros/protocol.cgi?id=4

Integration via Gamma Radiation -- http://www.biochem.northwestern.edu/ibis/morimoto/research/Protocols/IX.%20C.%20elegans/E.%20Genetics/1.%20Integration%20via%20gamma.pdf

MMS integration

Joe’s mRNA Prep -- http://www.k-state.edu/hermanlab/protocols/joes_mrna_prep.htm

F2 Mutant Screen - non "clonal" -- http://cobweb.dartmouth.edu/cgi-bin/cgiwrap/~ambros/protocol.cgi?id=27

C. elegans RNA Prep Protocol

Expression Lab - RNAi Protocol

Cosuppression in C. elegans – Ketting, et al. pdb ...

EMS mutagenesis

A Guide to Genetic Mapping in C. elegans-- http://uwacadweb.uwyo.edu/UWmolecbio/

Introduction of Double-Stranded RNA in C. elegans by Injection ...

Introduction of Double-Stranded RNA in C. elegans by Feeding ...

Introduction of Double-Stranded RNA in C. elegans by Soaking ...

Isolation of RNA from C. elegans -- Ketting et al. pdb ...

Mapping C. elegans Mutants using STS Polymorphisms -- http://cobweb.dartmouth.edu/cgi-bin/cgiwrap/~ambros/protocol.cgi?id=32

Preparation of C. Elegans Genomic DNA --C

Procedure: --Single Worm PCR

C. elegans gene knockout protocol - new

EMS Mutagenesis Of C. Elegans

Model Organisms/C. Elegans Protocols

C.elegans Gene Knockout Protocol by TMP/UV Method

Nature Protocols: RNAi screens in Caenorhabditis elegans in a 96 ...

Caenorhabditis briggsae methods *

TOTAL RNA-ISOLATION FROM C. ELEGANS ...

RNAi in C. elegans.pdb ...

Gene Knockout Protocol by TMP/UV Method

Identification of 1088 New Transposon Insertions of Caenorhabditis ...

Springer Protocols: Abstract: Intracellular Ca2+ Imaging in C. elegans

C. Elegans methods Floatation, hypochlorite treatment; liquid Culture, RNA Prep ...

Genes misregulated in C. elegans deficient in Dicer, RDE-4, or RDE .

Online Text for “Improving the Caenorhabditis elegans Genome Annotation ...

Homologous gene targeting in Caenorhabditis elegans by biolistic ...

Cloning and characterization of the C.elegans histidyl- tRNA ...

Isolating Mutants of the Nematode Caenorhabditis elegans That Are ...

MultiSite Gateway Recombination System Using a C. Elegans ...

Methods - C. elegans - Cenix Protocol for Production of large Scale dsRNA ...

Towards a mutation in every gene in Caenorhabditis elegans ...

Mos as a tool for genome-wide insertional mutagenesis in C. Elegans ...

IngentaConnect Shotgun cloning of transposon insertions in the Genome of C. Elegans ...

Preparation of C. Elegans Genome DNA - C

Genetics and Neurobiology of C. elegans - Publications

Shotgun cloning of transposon insertions in the genome of C. Elegans...

Identification of 1088 New Transposon Insertions of Caenorhabditis Elegans ...

Genome-wide RNAi screening in Caenorhabditis elegans

Biolistic transformation in C. elegans using unc-119 rescue

Discovery of novel alternatively Spliced C. Elegans Transcripts by Computational Analysis of Sage Data...

Using Stress Resistance to Isolate Novel Longevity Mutations in ...

Biomedical Pathways

2010-12-25T05:57:00.001-08:00

BIOCHEMICAL PATHWAY DIAGRAMS

Stages of Catabolism

Calvin Cycle Flow Diagram

Glycolysis

Glycolysis Enzyme Mechanisms

Glyoxylate Cycle

Aerobic Glucose Metabolism

Fatty Acid Oxidation Reactions

Glycolysis/Gluconeogenesis Pathways

Fatty Acid Synthase Complex

Gluconeogenesis in the Liver

Fatty Acid Biosynthesis Reactions

Pentose Phosphate Pathway

Pyruvate-Malate Cycle

Glycogen Cascade Control

Fatty Acid Regulation

Pyruvate DH Complex Reactions

Kreb's Urea Cycle

Kreb's TCA Cycle

Basic Folate Metabolism Pathways

Kreb's TCA Cycle Intermediate Metabolism

Purine Biosynthesis

Malate-Aspartate Shuttle

Purine Degradation

(Sourece: Richard A. Paselk, Humboldt State University)

Other Resources of Biochemical Pathway

Metabolic pathway - Wikipedia, the free encyclopedia

In biochemistry, a metabolic pathway is a series of chemical reactions occurring within a cell. In each pathway, a principal chemical is modified by ...
en.wikipedia.org/wiki/Metabolic_pathway

Image results for biochemical pathways

ExPASy - Biochemical Pathways and Pathway Map

Digitized version of wall charts courtesy Boehringer Mannheim et al, divided into Metabolic Pathways and Cellular and Molecular Processes, maintained by the ...
www.expasy.ch/cgi-bin/show_thumbnails.pl

Pathway Tools Query Page

Each dataset contains classification hierarchies for pathways, for reactions ( the enzyme nomenclature system), for compounds, and for genes. ...
pathway.yeastgenome.org/biocyc/

Pathways

Pathways is provided an an ancillary to General, Organic and Biochemistry and Concepts of Biochemistry. Please send any comments or suggestions to Jim Hardy ...
ull.chemistry.uakron.edu/Pathways/index.html

Yeast Biochemical Pathways

Yeast Biochemical Pathways are created using the Pathway tools software developed by Peter Karp and his colleagues at SRI International. ...
www.yeastgenome.org/biocyc/

T cell purification from splenocytes

2010-12-25T05:56:00.002-08:00

Procedure:

Remove the spleen.

Grind the spleen with the flat end of a syringe in 5 ml of RPMI on a 100 mm culture dish.

Pass through all cells through a cell strainer into a 50 ml tube. Wash the cell culture dish with 5 ml of RPMI twice and combine all cells.

Spin down the cells and remove the supernatant. Re-suspend the cell pellet into 5 ml of red blood cell lysis buffer followed by incubation at RT for 2 min. Dilute cells with 45 ml of RPMI or 1X PBS.

Spin down cells and re-suspend the cell pellet in 1 ml of RPMI/10% FCS. Incubate the cells at RT for 30 min.

Purify T cells with a sterile Nylon column. Wet the column with 5 ml of RPMI / 10% FCS followed by another 5 ml wash. Leave 1 ml of medium on the top of the column.

Incubate the column at 37oC for ~ 1 hour.

Open stop cock and allow media to run through column. Add 2 ml media and allow this to run through the column. Add 1 ml of splenocytes (1-2 x 108 cells). Allow cells to enter column. Add another 1 ml of media and allow to enter column. Cover the column with 1 ml media and incubate at 37oC for one hour.

Collect the T cells by washing column with 10 ml of media. Do not plunge, as this removes B cells.

Spin and re-suspend the cells and count them.

To 1 ml of cells (1-6 x107) add 8 µl (8 µg) of anti-MHCII and / or 8 µl (4 µg) of anti-CD8.

Incubate on ice for 30 min. Add 9 ml of PBS or RPMI. Spin and re-suspend the cells into 1 ml of complement. Incubate for 30 min at 37oC. Commercially available complement reagent is prepared by re-suspending the lyophilyzed pellet in 1 ml of cold H2O. This should be diluted in 9 ml of RPMI.

After incubation, add 9 ml of RPMI or PBS. Repeat antibody-complement cycle once more. Wash the cells with RPMI and count.

Other Protocols:

B Cell Isolation

Dynal Biotech (Invitrogen) - Dynabeads ( T Cell Isolation Procedure)

Fractionation of T and B Cells Using Magnetic Beads Protocol –

Isolation of Ly-1+/CD5+ B Cells by Cell Sorting Protocol –

Isolation of Resting B Lymphocytes from Sixteen Mouse Spleens Protocol –

Isolation of Resting B Lymphocytes Protocol –

Lymphocyte Isolation and Culture Protocol –

MagCellect Mouse CD3 T Cell Isolation Kit

Nature Protocols: Generation of multivirus-specific CTLs

Protocol: Immunofluorescence / confocal microscopy : B cells and T Cells.

R&D Systems - MagCellect* Mouse CD3+ T Cell Isolation ( manual)

The Immune System - in More Detail

Aseptic Technique

2010-12-25T05:56:00.001-08:00

• Protect eyes, mucous membranes, open cuts and wounds from contact with biohazard material

• Use gloves, goggles, mask, and protective clothing as necessary.

• Carry out all culturing operation in a laminar flow hood.

• Disinfect all surfaces prior to use with a disinfectant solution.

• Swab down the working surface liberally with 70% ethanol.

• Periodically spread a solution of 70% ethanol over the exterior of gloves to minimize contamination. Replace them if torn.

• In case of any spill, spread a solution of 70% alcohol and swab immediately with non-linting wipes.

• Discard gloves after use and do not wear them when entering any other lab area. Bring into the work area only those items needed for a particular procedure.

• Leave a wide clear space in the center of the hood (not just the front edge) to work on. Do not clutter the area to prevent blockage of proper air flow and to minimize turbulence.

• Swab with 70% alcohol all glassware(medium bottles, beakers, etc.)before placing them inside the hood.

• Arrange the work area to have easy access to all of it without having to reach over one item to get at another (especially over an open bottle or flask).

• Use sterile wrapped pipettes and discard them after use into a biohazard waste container.

• Check that the wrapping of the sterile pipette is not broken or damaged.

• Inspect the vessels to be used:

a. T-flask – Must be free from visible contamination or breakage, or lack container

identification. The plastic covering the flask must be intact.

b. Bottles – Check for cracks, expiration dates.

c. Spinners flasks – Check for cracks, expiration dates, and proper assembly.

Discard any biohazard or contaminated material immediately.

Never perform mouth pipetting. Pipettor must be used.

When handling sterile containers with caps or lids, place the cap on its side if it must be laid on the work surface.

Make sure not to touch the tip of the pipette to the rim of any flask or sterile bottle.

Clean the work area when finished by wiping with 70% alcohol.

(Source: Openwetware)

Angiogenic Models-chorioallantoic membrane assay

2010-12-25T05:55:00.003-08:00

Biomacromolecules - CAM preparation

CAM preparation. IPW research units. Prof. D. Neri · Prof. H. Wunderli - Allenspach · Prof. U. Spichiger - Keller · Prof. H.P. Merkle · Prof. U. Quitterer ...
www.pharma.ethz.ch/institute_groups/biomacromolecules/protocols/cam

Chorioallantoic Membrane Vascular Assay (CAMVA)

Chorioallantoic Membrane Vascular Assay (CAMVA). Theory: Vascular damage (i.e. ghost vessels, capillary injection, or. hemorrhaging) of the chorioallantoic ...
www.iivs.org/pages/methods/CAMVAsummarysheet.pdf

The gelatin sponge–chorioallantoic membrane assay

The gelatin sponge–chorioallantoic membrane assay. Here we present a method for the quantification of angiogenesis and antiangiogenesis in the chick embryo ...
www.natureprotocols.com/2006/06/23/the_gelatin_spongechorioallant.php

THE CHICK CHORIOALLANTOIC MEMBRANE AS A MODEL TISSUE FOR SURGICAL Retinal Research Simulation.
factors in ocular tissues of normal rabbits on chorioallantoic. membrane assay. Tohoku J Exp Med 1988;154:63–70. 10. Prost M. Experimental studies on the ...
www.stanford.edu/~palanker/publications/CAM%20as%20retinal%20model.pdf

Chorioallantoic membrane assay: vascularized 3-dim.

PubMed is a service of the US National Library of Medicine that includes over 16 million citations from MEDLINE and other life science journals for ...
www.ncbi.nlm.nih.gov/pubmed/11547121

Assessment of Angiogenic Factors The Chick Chorioallantoic Membrane Assay.

Assessment of Angiogenic Factors The Chick Chorioallantoic Membrane Assay. By: Adam Jones3, Chisato Fujiyama3, Stephen Hague3, Roy Bicknell4 ...
www.springerprotocols.com/Abstract/doi/10.1385/1-59259-137-X:119

Chicken chorioallantoic membrane assay (Cytokines & Cells ...

Chicken chorioallantoic membrane assay. The following COPE entries contain this entry term or one of its hypertext synonyms: ...
www.copewithcytokines.de/cope.cgi?key=Chicken%20chorioallantoic%20membrane%20assay

THE SHELL-LESS CHIC CAM ASSAY: A MODEL FOR STUDYING MELANOMA

ANGIOTROPISM AND EXTRAVASCULAR MIGRATORY METASTASIS
http://www.med.miami.edu/mnbws/documents/06Lugassy.pdf

Chorioallantoic Membrane Vascular Assay - Alternative Ocular Irritation Testing

Chorioallantoic Membrane Vascular Assay Alternative Ocular Irritation. Day 7 - Vascular development of the Chorioallantoic Membrane. ASSAY HIGHLIGHTS: ...
www.camva.com/

The gelatin sponge-chorioallantoic membrane assay.

The gelatin sponge–chorioallantoic membrane assay ... quantification of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane (CAM) ...
www.nature.com/nprot/journal/v1/n1/abs/nprot.2006.13.html

angiogenic models

Classical angiogenesis assays include the chick chorioallantoic membrane, rabbit cornea assay, sponge implant models, matrigel plugs and conventional tumor ...
www.med.unibs.it/~airc/sandra/models.html

Evaluating Compounds Affecting Angiogenesis.indd

ASSAY. In a typical angiogenesis evaluation using the CAM assay,. fertilized chick eggs are incubated at 37°C and specific. humidity (60%) for 3 to 4 days. ...
www.sri.com/biosciences/pdf/EvaluatingCompoundsAffectingAngiogenesis.pdf

All Mouse Models Federated Database, NIH

2010-12-25T05:55:00.001-08:00

34 Records Found

Note: Please click on the Mouse Name for more information


Note: Please click on the Mouse Name for more information
Mouse Name	Gene Name		Mutation Type	Manipulation Type
Aprt null	adenine phosphoribosyl transferase [APRT]		Null	targeted
ATM null	ataxia telangiectasia mutated (includes complementation groups A, C and D) [ATM]		Null	targeted
Brca1 cre Null	breast cancer 1, early onset [BRCA1]		loxP Sites	targeted
creK5			None selected	transgenic
CycD1bK5(A870G/exon5)	cyclin D1 (PRAD1: parathyroid adenomatosis 1) [CCND1/Cyclin D1]		None selected	transgenic
Cyclin D1b tg	cyclin D1 (PRAD1: parathyroid adenomatosis 1) [CCND1/Cyclin D1]		None selected	transgenic
Cyclin Db1 (A870G/exon 5)	cyclin D1 (PRAD1: parathyroid adenomatosis 1) [CCND1/Cyclin D1]		None selected	transgenic
DNAjC3(tm-gfp)	DnaJ (Hsp40) homolog, subfamily C, member 3 [DNAJC3]		Null	targeted
Exo 1 null	exonuclease 1 [EXO1]		Null	targeted
Fen1 null	flap structure-specific endonuclease 1 [FEN1]		Null	targeted
Gal4 E2F1	E2F transcription factor 1 [E2F1]		None selected	transgenic
Ku70 null	Thyroid autoantigen 70kDa, Ku antigen [G22P1]		Null	targeted
MBD4 null	methyl-CpG binding domain protein 4 [MBD4]		Null	targeted
MCAT Tg	Mitochondrial - Targeted Catalase [MCAT]		None selected	transgenic
MIh1 null	mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) [MLH1]		Null	targeted
Msh2 (G674A) ATPase	mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli) [MSH2]		Null	targeted
Msh2 null	mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli) [MSH2]		Null	targeted
Msh6(T1217D)	mutS homolog 6 (E. coli) [MSH6]		None selected	targeted
P53(R270H)	tumor protein p53 (Li-Fraumeni syndrome) [TP53]		Point Mutant	targeted
P53.R270H/balancer-Cre	tumor protein p53 (Li-Fraumeni syndrome) [TP53]		Point Mutant	targeted
P53.R270H/K14Cre	tumor protein p53 (Li-Fraumeni syndrome) [TP53]		Point Mutant	targeted
P53.R270H/Wap-Cre	tumor protein p53 (Li-Fraumeni syndrome) [TP53]		Point Mutant	targeted
P53(S389A)	tumor protein p53 (Li-Fraumeni syndrome) [TP53]		Point Mutant	targeted
PCNA Pol130-104	proliferating cell nuclear antigen [PCNA]		None selected	targeted
Plap-Fs			Null	transgenic
Plap-ti			Null	transgenic
POLdelta1 (D400A)	polymerase (DNA directed), delta 1, catalytic subunit [POLD1]		Point Mutant	targeted
PRKR	protein kinase, interferon-inducible double stranded RNA dependent [PRKR]		Point Mutant	transgenic
pUR 288-lacZ 30			Null	targeted
pUR 288-lacZ 60			Null	targeted
WRN K577M Tg	Werner syndrome [WRN]		Point Mutant	transgenic
			Point Mutant	transgenic
			Point Mutant	transgenic
XPV null		polymerase (DNA directed), eta [POLH]	Null	targeted