Biochemistry

Cloning Fact Sheet: questions and answers (2)

2011-06-30T22:31:00.000-07:00

What animals have been cloned?

Scientists have been cloning animals for many years. In 1952, the first animal, a tadpole, was cloned. Before the creation of Dolly, the first mammal cloned from the cell of an adult animal, clones were created from embryonic cells. Since Dolly, researchers have cloned a number of large and small animals including sheep, goats, cows, mice, pigs, cats, rabbits, and a gaur. All these clones were created using nuclear transfer technology.

Hundreds of cloned animals exist today, but the number of different species is limited. Attempts at cloning certain species have been unsuccessful. Some species may be more resistant to somatic cell nuclear transfer than others. The process of stripping the nucleus from an egg cell and replacing it with the nucleus of a donor cell is a traumatic one, and improvements in cloning technologies may be needed before many species can be cloned successfully.

Can organs be cloned for use in transplants?

Scientists hope that one day therapeutic cloning can be used to generate tissues and organs for transplants. To do this, DNA would be extracted from the person in need of a transplant and inserted into an enucleated egg. After the egg containing the patient's DNA starts to divide, embryonic stem cells that can be transformed into any type of tissue would be harvested. The stem cells would be used to generate an organ or tissue that is a genetic match to the recipient. In theory, the cloned organ could then be transplanted into the patient without the risk of tissue rejection. If organs could be generated from cloned human embryos, the need for organ donation could be significantly reduced.

Many challenges must be overcome before "cloned organ" transplants become reality. More effective technologies for creating human embryos, harvesting stem cells, and producing organs from stem cells would have to be developed. In 2001, scientists with the biotechnology company Advanced Cell Technology (ACT) reported that they had cloned the first human embryos; however, the only embryo to survive the cloning process stopped developing after dividing into six cells. In February 2002, scientists with the same biotech company reported that they had successfully transplanted kidney-like organs into cows. The team of researchers created a cloned cow embryo by removing the DNA from an egg cell and then injecting the DNA from the skin cell of the donor cow's ear. Since little is known about manipulating embryonic stem cells from cows, the scientists let the cloned embryos develop into fetuses. The scientists then harvested fetal tissue from the clones and transplanted it into the donor cow. In the three months of observation following the transplant, no sign of immune rejection was observed in the transplant recipient.

Another potential application of cloning to organ transplants is the creation of genetically modified pigs from which organs suitable for human transplants could be harvested . The transplant of organs and tissues from animals to humans is called xenotransplantation.

Why pigs? Primates would be a closer match genetically to humans, but they are more difficult to clone and have a much lower rate of reproduction. Of the animal species that have been cloned successfully, pig tissues and organs are more similar to those of humans. To create a "knock-out" pig, scientists must inactivate the genes that cause the human immune system to reject an implanted pig organ. The genes are knocked out in individual cells, which are then used to create clones from which organs can be harvested. In 2002, a British biotechnology company reported that it was the first to produce "double knock-out" pigs that have been genetically engineered to lack both copies of a gene involved in transplant rejection. More research is needed to study the transplantation of organs from "knock-out" pigs to other animals.

What are the risks of cloning?

Reproductive cloning is expensive and highly inefficient. More than 90% of cloning attempts fail to produce viable offspring. More than 100 nuclear transfer procedures could be required to produce one viable clone. In addition to low success rates, cloned animals tend to have more compromised immune function and higher rates of infection, tumor growth, and other disorders. Japanese studies have shown that cloned mice live in poor health and die early. About a third of the cloned calves born alive have died young, and many of them were abnormally large. Many cloned animals have not lived long enough to generate good data about how clones age. Appearing healthy at a young age unfortunately is not a good indicator of long-term survival. Clones have been known to die mysteriously. For example, Australia's first cloned sheep appeared healthy and energetic on the day she died, and the results from her autopsy failed to determine a cause of death.

In 2002, researchers at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, reported that the genomes of cloned mice are compromised. In analyzing more than 10,000 liver and placenta cells of cloned mice, they discovered that about 4% of genes function abnormally. The abnormalities do not arise from mutations in the genes but from changes in the normal activation or expression of certain genes.

Problems also may result from programming errors in the genetic material from a donor cell. When an embryo is created from the union of a sperm and an egg, the embryo receives copies of most genes from both parents. A process called "imprinting" chemically marks the DNA from the mother and father so that only one copy of a gene (either the maternal or paternal gene) is turned on. Defects in the genetic imprint of DNA from a single donor cell may lead to some of the developmental abnormalities of cloned embryos.

Should humans be cloned?

Physicians from the American Medical Association and scientists with the American Association for the Advancement of Science have issued formal public statements advising against human reproductive cloning. The U.S. Congress has considered the passage of legislation that could ban human cloning.

Due to the inefficiency of animal cloning (only about 1 or 2 viable offspring for every 100 experiments) and the lack of understanding about reproductive cloning, many scientists and physicians strongly believe that it would be unethical to attempt to clone humans. Not only do most attempts to clone mammals fail, about 30% of clones born alive are affected with "large-offspring syndrome" and other debilitating conditions. Several cloned animals have died prematurely from infections and other complications. The same problems would be expected in human cloning. In addition, scientists do not know how cloning could impact mental development. While factors such as intellect and mood may not be as important for a cow or a mouse, they are crucial for the development of healthy humans. With so many unknowns concerning reproductive cloning, the attempt to clone humans at this time is considered potentially dangerous and ethically irresponsible.

(Source: www.ornl.gov)

Cloning Fact Sheet: questions and answers (1)

2011-06-30T22:30:00.000-07:00

Introduction

The possibility of human cloning, raised when Scottish scientists at Roslin Institute created the much-celebrated sheep "Dolly" (Nature 385, 810-13, 1997), aroused worldwide interest and concern because of its scientific and ethical implications. The feat, cited by Science magazine as the breakthrough of 1997, also generated uncertainty over the meaning of "cloning" --an umbrella term traditionally used by scientists to describe different processes for duplicating biological material.

What is cloning? Are there different types of cloning?

When the media report on cloning in the news, they are usually talking about only one type called reproductive cloning. There are different types of cloning however, and cloning technologies can be used for other purposes besides producing the genetic twin of another organism. A basic understanding of the different types of cloning is key to taking an informed stance on current public policy issues and making the best possible personal decisions. The following three types of cloning technologies will be discussed: (1) recombinant DNA technology or DNA cloning, (2) reproductive cloning, and (3) therapeutic cloning.

Recombinant DNA Technology or DNA Cloning

The terms "recombinant DNA technology," "DNA cloning," "molecular cloning," and "gene cloning" all refer to the same process: the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid. The DNA of interest can then be propagated in a foreign host cell. This technology has been around since the 1970s, and it has become a common practice in molecular biology labs today.

Scientists studying a particular gene often use bacterial plasmids to generate multiple copies of the same gene. Plasmids are self-replicating extra-chromosomal circular DNA molecules, distinct from the normal bacterial genome (see image to the right). Plasmids and other types of cloning vectors were used by Human Genome Project researchers to copy genes and other pieces of chromosomes to generate enough identical material for further study.

To "clone a gene," a DNA fragment containing the gene of interest is isolated from chromosomal DNA using restriction enzymes and then united with a plasmid that has been cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined with its cloning vector in the lab, it is called a "recombinant DNA molecule." Following introduction into suitable host cells, the recombinant DNA can then be reproduced along with the host cell DNA.

Plasmids can carry up to 20,000 bp of foreign DNA. Besides bacterial plasmids, some other cloning vectors include viruses, bacteria artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Cosmids are artificially constructed cloning vectors that carry up to 45 kb of foreign DNA and can be packaged in lambda phage particles for infection into E. coli cells. BACs utilize the naturally occurring F-factor plasmid found in E. coli to carry 100- to 300-kb DNA inserts. A YAC is a functional chromosome derived from yeast that can carry up to 1 MB of foreign DNA. Bacteria are most often used as the host cells for recombinant DNA molecules, but yeast and mammalian cells also are used.

Reproductive Cloning

Reproductive cloning is a technology used to generate an animal that has the same nuclear DNA as another currently or previously existing animal. Dolly was created by reproductive cloning technology. In a process called "somatic cell nuclear transfer" (SCNT), scientists transfer genetic material from the nucleus of a donor adult cell to an egg whose nucleus, and thus its genetic material, has been removed. The reconstructed egg containing the DNA from a donor cell must be treated with chemicals or electric current in order to stimulate cell division. Once the cloned embryo reaches a suitable stage, it is transferred to the uterus of a female host where it continues to develop until birth.

Dolly or any other animal created using nuclear transfer technology is not truly an identical clone of the donor animal. Only the clone's chromosomal or nuclear DNA is the same as the donor. Some of the clone's genetic materials come from the mitochondria in the cytoplasm of the enucleated egg. Mitochondria, which are organelles that serve as power sources to the cell, contain their own short segments of DNA. Acquired mutations in mitochondrial DNA are believed to play an important role in the aging process.

Dolly's success is truly remarkable because it proved that the genetic material from a specialized adult cell, such as an udder cell programmed to express only those genes needed by udder cells, could be reprogrammed to generate an entire new organism. Before this demonstration, scientists believed that once a cell became specialized as a liver, heart, udder, bone, or any other type of cell, the change was permanent and other unneeded genes in the cell would become inactive. Some scientists believe that errors or incompleteness in the reprogramming process cause the high rates of death, deformity, and disability observed among animal clones.

Therapeutic Cloning

Therapeutic cloning, also called "embryo cloning," is the production of human embryos for use in research. The goal of this process is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease. Stem cells are important to biomedical researchers because they can be used to generate virtually any type of specialized cell in the human body. Stem cells are extracted from the egg after it has divided for 5 days. The egg at this stage of development is called a blastocyst. The extraction process destroys the embryo, which raises a variety of ethical concerns. Many researchers hope that one day stem cells can be used to serve as replacement cells to treat heart disease, Alzheimer's, cancer, and other diseases.

In November 2001, scientists from Advanced Cell Technologies (ACT), a biotechnology company in Massachusetts, announced that they had cloned the first human embryos for the purpose of advancing therapeutic research. To do this, they collected eggs from women's ovaries and then removed the genetic material from these eggs with a needle less than 2/10,000th of an inch wide. A skin cell was inserted inside the enucleated egg to serve as a new nucleus. The egg began to divide after it was stimulated with a chemical called ionomycin. The results were limited in success. Although this process was carried out with eight eggs, only three began dividing, and only one was able to divide into six cells before stopping.

How can cloning technologies be used?

Recombinant DNA technology is important for learning about other related technologies, such as gene therapy, genetic engineering of organisms, and sequencing genomes. Gene therapy can be used to treat certain genetic conditions by introducing virus vectors that carry corrected copies of faulty genes into the cells of a host organism. Genes from different organisms that improve taste and nutritional value or provide resistance to particular types of disease can be used to genetically engineer food crops. With genome sequencing, fragments of chromosomal DNA must be inserted into different cloning vectors to generate fragments of an appropriate size for sequencing.

If the low success rates can be improved (Dolly was only one success out of 276 tries), reproductive cloning can be used to develop efficient ways to reliably reproduce animals with special qualities. For example, drug-producing animals or animals that have been genetically altered to serve as models for studying human disease could be mass produced.

Reproductive cloning also could be used to repopulate endangered animals or animals that are difficult to breed. In 2001, the first clone of an endangered wild animal was born, a wild ox called a gaur. The young gaur died from an infection about 48 hours after its birth. In 2001, scientists in Italy reported the successful cloning of a healthy baby mouflon, an endangered wild sheep. The cloned mouflon is living at a wildlife center in Sardinia. Other endangered species that are potential candidates for cloning include the African bongo antelope, the Sumatran tiger, and the giant panda. Cloning extinct animals presents a much greater challenge to scientists because the egg and the surrogate needed to create the cloned embryo would be of a species different from the clone.

Therapeutic cloning technology may some day be used in humans to produce whole organs from single cells or to produce healthy cells that can replace damaged cells in degenerative diseases such as Alzheimer's or Parkinson's. Much work still needs to be done before therapeutic cloning can become a realistic option for the treatment of disorders.

Oxidase test

2011-06-30T22:29:00.000-07:00

Background: The oxidase test is a test used in microbiology to determine if a bacterium produces certain cytochrome c oxidases. It uses disks impregnated with a reagent such as N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) or N,N-Dimethyl-p-phenylenediamine (DMPD), which is also a redox indicator.

Strains may either be oxidase positive (OX+) or negative (OX-).

OX+

OX+ normally means that the b

acterium contains cytochrome c oxidase and can therefore utilize oxygen for energy production with an electron transfer chain. Typically the Pseudomonadaceae are OX+[citation needed] Another example is the preliminary identification of Neisseria and Moraxella genera, which are both oxidase positive, Gram-negative diplococci.

Many Gram-negative spiral curved rods are also oxidase positive, which includes Helicobacter pylori, Vibrio cholera, and Campylobacter jejuni.

Also Legionella pneumophila is oxidase positive. A trick to remember the most medical relevant bacteria is: "VIce President CHeNEy MOstly LEads" (Vibrio, Pseudomonas, Campylobacter, Helicobacter, Neisseria, Moraxella, and Legionella, respectively).

OX-

OX- normally

means that the bacterium does not contain cytochrome c oxidase and therefore cannot utilize oxygen for energy production with an electron transfer chain. Typically Enterobacteriaceae are OX-.

Procedures

Wet each disk with about 4 inoculating loops of de-ionized water.

Use a loop to aseptically transfer a large mass of pure bacteria to the disk.

Observe the disk for up to 3 minutes. If the area of inoculation turns dark blue to maroon to almost black, then the result is positive. If a color change does not occur within three minutes, the result is negative.

Alternatively, live bacteria cultivated on trypticase soy agar plates may be prepared using sterile technique with a single-line streak inoculation. The inoculated plates are incubated at 37°C for 24–48 hours to establish colonies. Fresh bacterial preparations should be used. After colonies have grown on the media, two-to-three drops of the reagent DMPD is added to the surface of each organism to be tested.

A positive test (OX+) will result in a color change to pink, through maroon and into black, within 10–30 seconds.

A negative test (OX-) will result in a light pink coloration or absence of coloration.

Lipid Monolayer Assay Protocol

2011-06-30T22:28:00.000-07:00

by Brian J. Peter and Matthew K. Higgins

Background

Lipid monolayers have been used for many years as templates for the formation of two-dimensional crystals of soluble proteins (reviewed in (1)) and, more recently, membrane proteins (2). The principle of the assay is that phospholipids on an aqueous droplet adopt a conformation in which the hydrophobic tails point towards the air while the hydrophilic head groups contact the solution. Proteins of interest interact with the head groups and are concentrated in a two-dimensional array. A hydrophobic electron microscope grid interacts with the lipid tails, allowing the monolayer to be removed from the droplet and studied in the electron microscope. The lipid monolayer simulates the inner leaflet of the plasma membrane, and can be used to reconstitute early stages of endocytosis. The formation of ordered clathrin assemblies can be observed using negative stain electron microscopy, and platinum shadowing can reveal the invagination of these coats. For examples, see references (3,4). A schematic of the technique and a gallery of images obtained with this technique can be viewed using the links, or at http://www2.mrc-lmb.cam.ac.uk/groups/hmm/epsin/EM/

Reagents

HKM buffer (25mM Hepes pH 7.4, 125mM potassium acetate, 5mM magnesium acetate, 1mM dithiothreitol).

Chloroform

methanol

Phosphatidylinositol and Phospatidylinositol-4,5-bisphosphate (Avanti polar lipids) and were dissolved to 1mg/ml in 3:1 chloroform: methanol. Cholesterol (also Avanti) was dissolved to 10mg/ml in Chloroform. Phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine (all from Sigma) were dissolved in chloroform to 10mg/ml. Lipid stocks were stored at -80C.

2% uranyl acetate (Biorad) with 0.0025% polyacrylic acid (Sigma) in water (see note 2)

Purified clathrin. Clathrin should be dialyzed into HKM buffer and centrifuged for 20 minutes at 100000 gmax (e.g., 45000 r.p.m. in a Beckman TLA100 rotor) immediately prior to use to remove aggregates.

Purified AP180, epsin, or other clathrin- and lipid-binding protein.

Equipment

Teflon block with 60 microliter wells allowing for side injection (see figure 1)

Carbon and collodion-coated gold electron microscopy grids (e.g., G204G from Agar Scientific, coated first with collodion or formvar, and then with a thin layer of evaporated carbon)

Humid chamber, or covered container with a wet paper towel inside

Forceps for handling EM grids—self-locking spring forceps are especially useful

Whatman filter paper or similar, for blotting EM grids.

Parafilm

Vacuum evaporator, 0.2mm diameter piece of platinum wire (TAAB Laboratories),1mm thick tungsten wire (also TAAB) (necessary for platinum shadowing)

Transmission electron microscope.

Procedure

1. Make up a lipid mixture containing 10% cholesterol, 40% PE, 40% PC and 10% PtdIns(4,5)P2 to a final concentration of 0.1mg/ml in a 19:1 mixture of chloroform: methanol (methanol is necessary to maintain PtdIns(4,5)P2 solubility). This mixture should be made on the day the monolayer is made. If stored, it should be stored under argon at –80 degrees in a glass vial with a glass or Teflon lid, for not longer than three days.

2. Arrange teflon block in humid chamber, and fill wells of teflon block with HKM buffer. Fill the wells with 40-60 microliters of buffer, such that the total volume in the well will be 60 microliters after injection of protein samples. See note 2.

3. Carefully pipet (or inject with Hamilton syringe) 1 microliter of lipid mixture on to the buffer in the well. As a negative control, inject pure chloroform without any lipid (this will test for lipid dependence of any structures seen, such as whether clathrin baskets form in solution instead of clathrin coats on the monolayer surface). See note 3.

4. Incubate at room temperature for 60 minutes. The chloroform should evaporate, leaving a monolayer of lipid on the surface of the buffer.

5. Carefully place one EM grid, carbon side down, onto the top of each buffer droplet. Grids should not be glow discharged before use as a hydrophobic carbon film is required to adhere to the hydrophobic lipid tails of the monolayer.

6. Gently inject proteins into the side injection well. Final protein concentrations in the well should be 0.5-2 micromolar for the AP180/epsin/adaptor protein, and 30-500 nanomolar for the clathrin. It is often useful to try several concentrations, to account for differences in protein activity.

7. Incubate 60 minutes at room temperature.

8. Prepare Uranyl acetate stain. Lay a fresh piece of Parafilm on the bench, and place two 30 microliter drops side by side on the Parafilm for each grid which will be stained. Lay out a piece of Whatman filter paper to blot buffer and stain from grids. Lay out a second piece of filter paper on which to set grids to air dry.

9. Gently inject approximately 30 microliters of buffer into the side injection port; this will raise the grid up above the surface of the teflon block. Immediately grab the grid with forceps and lift it vertically off of the droplet.

10. Blot the grid briefly by touching it to the filter paper, then touch it to the first stain droplet and blot immediately. Touch the grid to the second stain droplet, leave for 30 seconds, and blot briefly. This leaves a film of stain on the surface of the grid in which the protein is embedded. If the grids will be platinum shadowed, hold the grid to the filter paper for several seconds to ensure that the entire grid surface dries. Lay the grid on another piece of filter paper to dry.

11. For negative stain EM, grids can be examined in the EM immediately. They are also stable for several weeks, at least, at room temperature.

12. If platinum shadowing is required, set up the vacuum evaporator with a 2cm long piece of platinum wire coiled tightly round a piece of 1mm thick tungsten wire. Place the grids to be shadowed on a rotary platform at an angle of 10 to the line between the platform centre and the platinum coil. Create a vacuum in the evaporator. With a shield between the grids and the wire, turn on the current to the tungsten wire. When the platinum wire melts, remove the shield and the platinum will evaporate onto the grids. For rotary platinum shadowing, start rotation of the platform was immediately before removing the shield. For single angle shadowing, the platform can remain stationary during the evaporation. 1-2 minutes of evaporation is usually sufficient, but trials may need to be done to account for differences in evaporators.

Notes

This procedure will take approximately 3 hours. All reagents should be of the highest purity available, and buffers should be filtered before use.

1. The addition of polyacrylic acid helps to prevent the stain from precipitating and forming uranyl acetate crystals. But this is not essential, alternatively UrAc solutions can be clarified by filtration or centrifugation before use.

2. It is important to ensure that the surface of the droplet is either flat or slightly concave. Overfilling the teflon wells leads to a convex droplet surface, upon which the monolayer does not form properly. Also, filling the wells with too little volume (less than 35 microliters, or depending on the geometry of the block) can lead to an uneven surface near the side injection port.

3. Trace lipid contamination (e.g., PtdIns(4,5)P2) on the teflon block can result in misleading negative controls. Teflon block should be rinsed with hot water, then ethanol, and finally, soaked overnight in a mixture of chloroform/methanol to remove any protein or lipid residue. Hamilton syringes are also susceptible to trace lipid contamination.

References

1. Chiu W, Avila-Sakar AJ, Schmid MF. Electron crystallography of macromolecular periodic arrays on phospholipid monolayers. Adv Biophys. 1997;34:161-72.

2. Levy D, Mosser G, Lambert O, Moeck GS, Bald D, Rigaud JL. Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins.
J Struct Biol. 1999 Aug;127(1):44-52

3. Ford MG, Mills IG, Peter BJ, Vallis Y, Praefcke GJ, Evans PR, McMahon HT. Curvature of clathrin-coated pits driven by epsin. Nature. 2002;419(6905):361-6.

4. Ford MG, Pearse BM, Higgins MK, Vallis Y, Owen DJ, Gibson A, Hopkins CR, Evans PR, McMahon HT. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science. 2001;291(5506):1051-5.

Source:

http://www2.mrc-lmb.cam.ac.uk/groups/hmm/techniqs/mono.html

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, U.K. CB2 2QH

If there are questions or comments, contact bpeter@mrc-lmb.cam.ac.uk or Harvey: hmm@mrc-lmb.cam.ac.uk

Lipid-Protein Overlay Assay (Fat Blotting)

2011-06-30T22:27:00.000-07:00

Principle of the assay: This assay will test specific interactions between proteins and

immobilized lipids. It is essentially most similar to immunoblotting, with the added step of protein

binding preceding the antibody steps.

Special reagents and equipment:

Fatty Acid-Free (FAF) BSA Sigma Cat# A6003

Hybond C extra membrane Amersham Biosciences Cat # RPN2020E

Procedure:

1. Dilute lipids of interest in chloroform to equal concentration such that your desired amount

of lipid can be achieved in a total volume of 5 ul. *

2. Spot 5 ul of each dilution of lipid on to Hybond C extra membrane (mixed ester supported

nitrocellulose). **

3. Allow the membrane to dry at room temperature for at least 1 hr post-spotting. ***

4. Wet the membrane by floating on nano-purified water for 10 minutes, followed by

equilibration in buffer for 5 minutes (TBS-T (0.1% Tween-20)).

5. Block membrane in 3% FAF-BSA/TBS-T for 1 hr at room temperature. ****

6. Dilute your protein to 0.2 μg/ml in 3% FAF-BSA/TBS-T. Incubate membrane with protein

solution overnight at 4

C.*****.

7. Next morning: wash the membrane 6 times (5 minutes for each wash) with TBS-T.

8. Incubate with primary antibody in 3% FAF-BSA/TBS-T for 1 hr. at room temperature (do

not use nonfat dry milk, use the FAF-BSA throughout the entire procedure).******

9. Wash as in 7.

10. Incubate with secondary antibody in 3% FAF-BSA/TBS-T for 1 hr at room temperature.

11. Wash 12 times (5 minutes each wash) with TBS-T.

12. Visualize protein binding using enhanced chemiluminescence. Expose to film for 5

minutes for an initial trial and modify time as needed. .*******

Reference:

1. Stevenson, J.M., I.Y. Perera, and W.F. Boss, A phosphatidylinositol 4-kinase pleckstrin

homology domain that binds phosphatidylinositol 4-monophosphate. J Biol Chem, 1998.

273(35): p. 22761-7.

2. Dowler, S., et al., Identification of pleckstrin-homology-domain-containing proteins with novel

phosphoinositide-binding specificities. Biochem J, 2000. 351(Pt 1): p. 19-31.

3. Jones, J.A. and Y.A. Hannun, Tight Binding Inhibition of Protein Phosphatase-1 by

Phosphatidic Acid. SPECIFICITY OF INHIBITION BY THE PHOSPHOLIPID. J Biol Chem,

2002. 277(18): p. 15530-8. Jones JA, Hannun YA.Tight binding inhibition of protein phosphatase-1 by phosphatidic acid.

Specificity of inhibition by the phospholipid. J Biol Chem. 2002 May 3;277(18):15530-8. Epub

2002 Feb 20.

Author: Jeffrey Jones (modified by Bill Wu)

Your email: wub@musc.edu

Date: Dec-2009

Hop DNA Extraction Protocol

2011-06-30T22:26:00.000-07:00

1. Obtain an adequate amount (~ 1g) of fresh hop leaves and crush them with liquid Nitrogen and a small amount of Carborundum powder (fine 320 grit).

2. Assume 90% of mass is water weight.

3. Add 3.3 ml of buffer per gram of wet (16 ml per gram of dried) hop leaves, and incubate for 1 to 4 hours at 60-65°C.

4. Transfer 900 μl into fresh tube

5. add 600 μl of 24:1 CHCl3:octanol and invert gently (do NOT vortex!).

6. Centrifuge at 5000g for 10 minutes.

7. Transfer supernatant (800 μl) into new 2-ml tube.

8. Add 5μl of RNAase and incubate at 37°C for 30 minutes (or more).

9. Add 0.6 volumes Isopropanol and mix gently by inverting the tubes. Check for DNA precipitation.

10. Spin down for 10 min. at RT.

11. Add 500 μl wash buffer and incubate 10 min. at RT.

12. Carefully remove wash buffer. Don't lose DNA pellet!

13. Briefly centrifuge to collect pellet at bottom of tube - remove any remaining wash buffer.

14. Dry pellet at RT or 50°C to speed up.

15. Add 100 μl ddH2O to dissolve DNA.

16. Store at -20°C until needed.

17. Run electrophoresis for analysis.

Prepared solutions

Buffer: 100 ml: 50 mM Tris/HCl (ph 8.0), 1.8 M NaCl, 50 mM EDTA. Then add 10 mg/ml of CTAB ( 200 mg per 20 ml buffer, final conc. = 1%) and 1 μl/ml 2-mercaptoethanol (20 μl to 20 ml buffer; final conc. = 0.1%).

Wash buffer 100 ml: 200 μl 5M NH4OAc (final conc. = 10 mM), 76.0 ml abs. ethanol (final conc. = 76%), and 23.8 ml of sterilized water.

Bioprotocols: Hop DNA Extraction Protocol

25 Dec 2010 ... Hop DNA Extraction Protocol. 1. Obtain an adequate amount (~ 1g) of fresh hop leaves and crush them with liquid Nitrogen and a small amount ...
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Robust CTAB-activated charcoal protocol for plant DNA extraction
author：M KRIŽMAN - 2006
Dried hop cones were obtained from the experimental fields (yield 2005) .... Modification of a CTAB DNA extraction protocol for plants ...
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Isolation of plant DNA: A fast, inexpensive, and reliable method

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Von Kossa Stain for Calcium Detection

2010-11-09T05:31:00.001-08:00

The method was originally developed by von Kossa, and underwent several modifications. The stain principle is a precipitation reaction in which silver ions react with phosphate (not calcium) in the presence of acidic material. Photochemical degradation of silver phosphate to silver then occurs under light illumination.

Additional methods need to be employed to confirm the presence of calcium and hydroxyapatite, such as Alizarin Red, which detects calcium deposits.

VON KOSSA Stain Procedure:

1. Sections to water.

2. Place sections in 1% silver nitrate solution under reasonable light for 30-60 minutes.

3. Wash in distilled water.

4. Treat with 3% sodium thiosulphate for 3-5 minutes.

5. Wash in water.

6. Counterstain with van Gieson for 3-5 minutes.

7. Wash in alcohol.

8. Clear .

9. Mount sections

RESULTS:

Mineralised bone black Osteoid red

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www.bdbiosciences.com/external_files/dl/doc/tech.../TB444.pdf

von Kossa staining alone is not sufficient to confirm that ...
Numerous techniques are currently used to characterize biological mineralization in intact tissues and cell cultures; the von Kossa staining method, ...
www.ncbi.nlm.nih.gov/pubmed/12724828

The von Kossa reaction for calcium deposits: silver lactate ...
We have demonstrated that positive von Kossa stain- ing correlates well with calcifications in... cultures the von Kossa stain usually yielded identifiable ...
www.springerlink.com/index/k782p815tn252t39.pdf

Re: [Histonet] von kossa stain

27 Jan 2004 ... My understanding of the von Kossa stain is that it is NOT specific for calcium. Rather, the silver nitrate in the staining solution (silver ...
www.histosearch.com/histonet/.../Re.Histonetvonkossastain.html

VON KOSSA STAIN KIT PROCEDURE
VON KOSSA STAIN KIT PROCEDURE. Item# KT 028. KIT. 100ml. Nuclear Fast Red Stain. 100ml. 5% Silver Nitrate. COMPONENTS. 100ml. 5% Sodium Thiosulfate ...
search.cosmobio.co.jp/cosmo_search_p/..._/KT028.20060119.pdf

von Kossa stain - Definition

von Kossa stain definition from the mondofacto online medical dictionary.
www.mondofacto.com/facts/dictionary?von+Kossa+stain

Von Kossa Staining Alone Is Not Sufficient to Confirm that ...
and cell cultures; the von Kossa staining method, elec- tron microscopic analysis (EM), ....However, the von Kossa stain is being used with greater ...
www.springerlink.com/index/TWUFXJY1VTHUVYR5.pdf

von Kossa and Alcian Blue Staining Protocol for Calcium and ...

Combination of von Kossa and Alcian Blue staining will make both calcium and mucosubstances visible on the same tissue sections. ...
www.ihcworld.com/_...stains/von_kossa.alcian_blue.htm

Histology Lab: VON KOSSA

Histotechnology Technical methods. Stain for calcium ... VON KOSSA. 1. Sections to water. 2. Place sections in 1% silver nitrate solution under ultra- ...
www.nottingham.ac.uk/pathology/protocols/vonkoss.html

Von kossa stain: Definition with Von kossa stain Pictures and Photos

Definition of Von kossa stain with photos and pictures, translations, sample usage, and additional links for more information.
www.lexic.us/definition-of/von_kossa_stain

Stain Expert™ - Your Stains & Reagents Information Center

Calcium Stain - Von Kossa. Celestine Blue Method - Lendrum. Copper Stain (Rubeanic Acid). Cresyl Violet Acetate Method for Helicobacter sp. ...
www.sigmaaldrich.com

JoVE: Undecalcified Bone Preparation for Histology ...
8 Jan 2010 ... An identical protocol or rack staining is necessary for ... Von Kossa. Place in silver nitrate solution and expose to strong light until ...
www.jove.com/index/details.stp?id=1707

Isolation of human multipotent mesenchymal stem cells from second ...
We developed a novel two‐stage culture protocol for isolating MSCs from amniotic .... (d) Osteogenic differentiation was demonstrated by von Kossa staining. ...
humrep.oxfordjournals.org/content/19/6/1450.full

BioMed Central | Full text | Isolation of mesenchymal stem cells ...
30 May 2007 ... Von Kossa staining of calcium-phosphate deposits was performed. ... (1999) [40] and a slightly modified protocol of the one reported by ...
www.biomedcentral.com/1472-6750/7/26

Fwd: [Histonet] Question on Von Kossa

Of course, as with any silver stain use acid cleaned glassware and gloves ... Does anyone have a good Von Kossa stain protocol that they would not mind ...
www.mail-archive.com/histonet@lists...edu/msg00672.html

Von Kossa Stain for Calcium Detection

2010-11-09T05:31:00.000-08:00

Additional methods need to be employed to confirm the presence of calcium and hydroxyapatite, such as Alizarin Red, which detects calcium deposits.

VON KOSSA Stain Procedure:

1. Sections to water.

2. Place sections in 1% silver nitrate solution under reasonable light for 30-60 minutes.

3. Wash in distilled water.

4. Treat with 3% sodium thiosulphate for 3-5 minutes.

5. Wash in water.

6. Counterstain with van Gieson for 3-5 minutes.

7. Wash in alcohol.

8. Clear .

9. Mount sections

RESULTS:

Mineralised bone black Osteoid red

Other Protols for Von Kossa

von Kossa Staining Protocol for Calcium

Von Kossa Stain - Wikipedia, the free encyclopedia

Von Kossa Stain. From Wikipedia, the free encyclopedia. Jump to: navigation, search. Thevon Kossa stain is used to quantify mineralization in cell culture ...
en.wikipedia.org/wiki/Von_Kossa_Stain

Renal Fellow Network: the von Kossa Stain for Acute Phosphate ...

Re: [Histonet] von kossa stain

von Kossa stain - Definition

von Kossa stain definition from the mondofacto online medical dictionary.
www.mondofacto.com/facts/dictionary?von+Kossa+stain

von Kossa and Alcian Blue Staining Protocol for Calcium and ...

Combination of von Kossa and Alcian Blue staining will make both calcium and mucosubstances visible on the same tissue sections. ...
www.ihcworld.com/_...stains/von_kossa.alcian_blue.htm

Histology Lab: VON KOSSA

Von kossa stain: Definition with Von kossa stain Pictures and Photos

Definition of Von kossa stain with photos and pictures, translations, sample usage, and additional links for more information.
www.lexic.us/definition-of/von_kossa_stain

Stain Expert™ - Your Stains & Reagents Information Center

Calcium Stain - Von Kossa. Celestine Blue Method - Lendrum. Copper Stain (Rubeanic Acid). Cresyl Violet Acetate Method for Helicobacter sp. ...
www.sigmaaldrich.com

Fwd: [Histonet] Question on Von Kossa

Telomere and telomerase assay (2)

2010-01-15T10:05:00.000-08:00

Relative Gene Expression in Normal and Tumor Tissue by Quantitative RT-PCR

Author(s): Dennis S. Salonga, Kathleen D. Danenberg, Jean Grem, Ji Min Park, Peter V. Danenberg

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:83; Page Range: 83-98

Abstract | Full Text | PDF (138K)

Quantitative Detection of Telomerase Components by Real-Time, Online RT-PCR Analysis with the LightCycler

Author(s): Thomas Emrich, Sheng-Yung Chang, Gerlinde Karl, Birgit Panzinger, Chris Santini

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:99; Page Range: 99-108

Abstract | Full Text | PDF (98K)

Standard TRAP Assay

Author(s): Angelika M. Burger

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:109; Page Range: 109-124

Abstract | Full Text | PDF (131K)

Stretch PCR Assay

Author(s): Jun-ichi Nakayama, Fuyuki Ishikawa

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:125; Page Range: 125-136

Abstract | Full Text | PDF (110K)

Fluorescent Detection of Telomerase Activity

Author(s): Wade K. Aldous, Amber J. Marean, Mary J. DeHart, Katherine H. Moore

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:137; Page Range: 137-146

Abstract | Full Text | PDF (99K)

Nonradioactive Detection of Telomerase Activity Using a PCR-ELISA-Based Telomeric Repeat Amplification Protocol

Author(s): Thomas Emrich, Gerlinde Karl

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:147; Page Range: 147-158

Abstract | Full Text | PDF (123K)

In Situ TRAP Assay Detection of Telomerase Activity in Cytological Preparations

Author(s): Kazuma Ohyashiki, Junko H. Ohyashiki

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:159; Page Range: 159-164

Abstract | Full Text | PDF (143K)

Biotinylated Primer for Detecting Telomerase Activity Without Amplification

Author(s): Daekyu Sun

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:165; Page Range: 165-171

Abstract | Full Text | PDF (81K)

Whole-Cell and Microcell Fusion for the Identification of Natural Regulators of Telomerase

Author(s): Henriette Gourdeau, Marsha D. Speevak, Lucie Jetté, Mario Chevrette

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:173; Page Range: 173-195

Abstract | Full Text | PDF (145K)

Screening with COMPARE Analysis forTelomerase Inhibitors

Author(s): Imad Naasani, Takao Yamori, Takashi Tsuruo

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:197; Page Range: 197-207

Abstract | Full Text | PDF (125K)

Telomerase as a Therapeutic Target: Therapeutic Potential of Telomerase Inhibitors

Author(s): John A. Double

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:209; Page Range: 209-216

Abstract | Full Text | PDF (71K)

Methods of Telomerase Inhibition
Additionally, methods are outlined in this chapter for determining the effectiveness oftelomerase inhibition through TRAP assays or assessment of telomere ...
www.ncbi.nlm.nih.gov/pmc/articles/PMC2423206/

Telomere and telomerase assay (1)

2010-01-15T10:04:00.002-08:00

A telomere is a region of repetitive DNA at the end of a chromosome, which protects the end of the chromosome from deterioration. Its name is derived from the Greek nouns telos (τἐλος) "end" and merοs (μέρος, root: μερ-) "part".

Russian theorist Alexei Olovnikov was the first to recognize (1971) the problem of how chromosomes could replicate right to the tip, as such was impossible with replication in a 5' to 3' direction. To solve this and to accommodate Leonard Hayflick's idea of limited somatic cell division, Olovnikov suggested that DNA sequences would be lost in every replicative phase until they reached a critical level, at which point cell division would stop.

During cell division, the enzymes that duplicate the chromosome and its DNA cannot continue their duplication all the way to the end of the chromosome. If cells divided without telomeres, they would lose the ends of their chromosomes, and the necessary information they contain. (In 1972, James Watson named this phenomenon the "end replication problem".) The telomeres are disposable buffers blocking the ends of the chromosomes and are consumed during cell division and replenished by an enzyme, the telomerase reverse transcriptase.

Elizabeth Blackburn compared telomeres to the aglets (tips) on the ends of shoelaces that keep them from fraying.

In 1975–1977, Blackburn, working as a postdoctoral fellow at Yale University with Joseph Gall, discovered the unusual nature of telomeres, with their simple repeated DNA sequences composing chromosome ends. Their work was published in 1978. The telomere shortening mechanism normally limits cells to a fixed number of divisions, and animal studies suggest that this is responsible for aging on the cellular level and sets a limit on lifespans. Telomeres protect a cell's chromosomes from fusing with each other or rearranging—abnormalities which can lead to cancer—and so cells are normally destroyed when their telomeres are consumed. Most cancers are the result of "immortal" cells which have ways of evading this programmed destruction.

Elizabeth Blackburn, Carol Greider, and Jack Szostak were awarded the 2009 Nobel Prize in Physiology or Medicine for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase.

Telomeres and telomerase: biological and clinical importance ...
(15) developed an assay for testing telomerase activity in cell extracts. Based on identification of telomerase mechanisms and properties, the telomere ...
www.clinchem.org/cgi/content/full/43/5/708

Telomere Length Analysis and In Vitro Telomerase Assay.

1 Introduction; 2 Materials and Methods; 2.1 Telomere Measurement; 2.2 Telomere Cloning; 2.3 TRAP Telomerase Assay; 3 Notes; References. Browse by Subject ...
www.springerprotocols.com/Abstract/doi/10.../1-59259-434-4:123

Facts about Telomeres and Telomerase

The following section will teach you the basics of telomeres and telomerase. It will also introduce you to the potential applications of current telomerase ...
www4.utsouthwestern.edu/cellbio/shay.../sw_facts.html

TRAP–LIG, a modified telomere repeat amplification protocol assay to quantitate telomerase inhibition by small molecules.
Its major function is to maintain the length of telomeric DNA by synthesizing telomeric DNA repeats, and its enzymatic activity is assayed by means of the ...
linkinghub.elsevier.com/retrieve/pii/S0003269708002960

Telomeres and Telomerase: The Cellular Timekeepers

The telomeres appear to be the cellular timekeepers, but are they related to human aging?
www.senescence.info/telomeres.html

Effect of Telomere and Telomerase Interactive Agents on Human Tumor and Normal Cell Lines.
The cytotoxic effect of telomere and telomerase interactive agents against normal human marrow cells in the colony-forming assay showed the same range of ...
clincancerres.aacrjournals.org/content/6/3/987.full

Cell Research - Telomere and telomerase in oncology
Shortening of the telomeric DNA at the chromosome ends is presumed to limit the lifespan of... Telomere, telomerase, cancer, telomerase assay, inhibitor ...
www.nature.com

Telomeres and telomerase — Genes & Development
Cdc13p (gray) and Est1 (purple) may recruit telomerase to the telomeric 3′ end in ..... (1995) An in vitro assay for Saccharomyces telomerase requires EST1. ...
genesdev.cshlp.org/content/13/18/2353.full

Telomerase Assay in Renal Cancer

Bacchetti, S. and Counter, C. M. (1995) Telomeres and telomerase in human cancer. ...quantitative nonisotopic assay for telomerase activity in humantumors. ...
www.springerprotocols.com/Abstract/doi/10.1385/1-59259-144-2:05

Introduction to Telomeres and Telomerase

Author(s): Michael C. Bibby

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:01; Page Range: 1-12

Abstract | Full Text | PDF (81K)

Detection of Chromosome Ends by Telomere FISH

Author(s): Harry Scherthan

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:13; Page Range: 13-31

Abstract | Full Text | PDF (117K)

Telomere Length Distribution: Digital Image Processing and Statistical Analysis

Author(s): Jean-Patrick Pommier, Laure Sabatier

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:33; Page Range: 33-63

Abstract | Full Text | PDF (351K)

Analysis of Telomerase RNA Gene Expression by In Situ Hybridization

Author(s): W. Nicol Keith, Joseph Sarvesvaran, Martin Downey

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:65; Page Range: 65-81

Abstract | Full Text | PDF (132K)

Protein Purification Protocols (2)

2010-01-15T10:04:00.001-08:00

Related Articles

1. Expression and Purification of Soluble and Correctly Folded Bovine β-Lactoglobulin Variants A and B in Escherichia coli for NMR Studies
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 16 December 2009
Komala Ponniah, Trevor S. Loo, Patrick J.B. Edwards, Steven M. Pascal, Geoffrey B. Jameson, ...

2. Expression of soluble and functional human neonatal Fc receptor in Pichia pastoris
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 16 December 2009
Chang-Han Lee, Dong-Ki Choi, Hye-Ji Choi, Moo-Young Song, Yong-Sung Kim

3. Formate-nitrate transporters: optimisation of expression, purification and analysis of prokaryotic and eukaryotic representatives
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 16 December 2009
Katherine S.H. Beckham, Jane A. Potter, Shiela E. Unkles

4. Streamlined, automated protocols for the production of milligram quantities of untagged recombinant human cyclophilin-A (hCypA) and untagged human proliferating cell nuclear antigen (hPCNA) using ÄKTAxpress™
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5. Expression, purification and primary crystallographic study of human androgen receptor in complex with DNA and coactivator motifs
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6. Purification and characterization of recombinant CH3 domain fragment of the CREB-binding protein
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7. Establishment of a simple and rapid method to screen for strong promoters in Bacillus subtilis
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8. An Improved Procedure for the Purification of the Escherichia coli RNA Polymerase Omega Subunit
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Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 4 December 2009
Catherine E. Vrentas, Tamas Gaal, Richard R. Burgess, Richard L. Gourse

9. Establishment of a simple and rapid method to screen for strong promoters in Bacillus subtilis
Publication year: 2009
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10. An Improved Procedure for the Purification of the Escherichia coli RNA Polymerase Omega Subunit
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 4 December 2009
Catherine E. Vrentas, Tamas Gaal, Richard R. Burgess, Richard L. Gourse

Protein Purification (from Wikipedia, the Free Encyclopedia)

Contents

1 Purpose

2 Strategies

3 Evaluating purification yield

4 Purification of a tagged protein

5 Methods of protein purification

6 Extraction

7 Precipitation and differential solubilization

8 Ultracentrifugation

9 Chromatographic methods

9.1 Size exclusion chromatography

9.2 Separation based on charge or hydrophobicity

9.3 Ion exchange chromatography

9.4 Affinity chromatography

9.4.1 Metal binding

9.4.2 Immunoaffinity chromatography

9.5 HPLC

10 Concentration of the purified protein

10.1 Lyophilization

10.2 Ultrafiltration

11 Analytical

11.1 Denaturing-Condition Electrophoresis

11.2 Non-Denaturing-Condition Electrophoresis

12 References

13 External links

Protein Purification Protocols (1)

2010-01-15T10:03:00.000-08:00

Protein Purification and Identification Protocols

Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterisation of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or a microbial culture. The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps exploit differences in protein size, physico-chemical properties and binding affinity.

1. ALKYL Aspartamide HIC Columns

2. Comparison of affinity tags for protein purification.

3. DEAE Column Preparation

4. DEAE column: FPLC

5. Green Fluorescent Protein Isolation (Gary J. Lindquester)

6. GST Fusion Protein Preparation

7. GST Fusion Protein Purification from Yeast

8. GST-Fusion Protein Purification

9. HeLa Cell Nuclei Preparation (John Garland, The Biochemistry and Molecular Biology Department of The James H. Quillen College of Medicine in Johnson City, TN)

10. HILIC and SEC Columns PolySULFOETHYL and PolyHYDROXYETHYL Columns

11. Isolation of Protein from Tissue (Reddy research laboratory, Neurological Sciences Institute, Oregon Health & Science University)

12. Making Affinity Columns

13. Making Conductivity Curves (Waters Lab)

14. PI-PLC Purification (Bjorkman Group, Howard Hughes Medical Institute at California Institute of Technology)

15. Polyaspartic Acid Weak Cation Exchange Columns

16. PolyGLYCOPLEX Complex Carbohydrates Columns

17. PolySULFOETHYL Aspartamide Strong Cation Exchange Columns

18. PolyWAX LP Weak Anion Exchange Columns

19. Preparation of Tonsil Lysate (Springer Lab, Harvard University)

20. Protein Extraction (Frank Bottone Jr.)

21. Protein Extraction from Tissues (Reddy research laboratory, Neurological Sciences Institute, Oregon Health & Science University)
Protein Isolation Protocols (Lamond Lab, University of Dundee)

22. Protein Isolation using Trizol (Frank Bottone Jr.)

23. Protein Purification

24. Protein Purification

25. Protein Purification

26. Protein Purification

27. Protein Purification (handbook)

28. Protein Purification (Michael Blaber)

29. Protein Purification (Michael Blaber)

30. Protein Purification (Michael Blaber)

31. protein purification (Pictures)

32. Protein Purification and Analysis Protocols and Applications Guide (Promega)

33. Protein Purification on 1G3 Column (Bjorkman Group, Howard Hughes Medical

34. Protein Purification Protocols (The Protein Expression and Purification Facility, EMBL)

35. Protein Sample Preparation & Protein Purification (Invitrogen)

36. Purificaiton of Protein by Affinity Chromatography (Krause Lab)

37. Purification of GST Fused Proteins

38. Purification of GST Fused Proteins

39. Purification of His-tagged proteins from Sf9 cells under native conditions

40. Purification of Proteins (NCBI)

41. Purification Scheme for Ubiquitin Expression Lysate (Sosnick Lab, University of Chicago)

42. Purification of Maltose Binding Proteins Fused Proteins

43. Purification of MBP (maLTose-Binding Proteins) Fused Proteins (Bowtell Lab

44. Removal of Nucleic Acids from Protein (Dr. Mario Lebendiker, The Hebrew University of Jerusalem)

45. RNA and protein extraction from the same tissue (Ivan J Delgado)

46. SDS Removal Guard Columns

47. Small Scale MBP-fusion Protein Purification (Dr. Mario Lebendiker, The Hebrew University of Jerusalem)

48. Subcellular Fractionation of Proteins (Bowtell Lab Manual, Peter MacCallum Cancer Institute)

49. Sucrose Density Gradient Fractionation of Yeast Membranes (Dohlman Lab)
Test Tube for Ion Exchange Chromatography (Dr. Mario Lebendiker, The Hebrew University of Jerusalem)

50. The Protein Purification Facility (Dr. Mario Lebendiker)

51. Tips for acid elution of peptides (Bjorkman Group, Howard Hughes Medical Institute at California Institute of Technology)

52. Whole Cell Protein Extracts (Morrisey Lab, University of Pennsylvania)
This is for both cytoplasmic and nuclear proteins.
Protein Purification - How to Purify Proteins.

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Protein Purification Handbook What is Biotechnology?Protein Engineering Methods What is a Protein?What are Enzymes?

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Stem Cell Research Progresses

2010-01-15T10:02:00.000-08:00

Stem Cells

Stem Cells Might Reverse Heart Damage from Chemo (12/30/2009, HealthDay)

Stem Cell Research Gets a Boost from Vitamin C (12/24/2009, HealthDay)

Most Stem Cells Used in Research Come from Whites (12/17/2009, HealthDay)

Adult Stem Cells Can Repair Damaged Heart (12/04/2009, HealthDay)

First Human Embryonic Stem Cell Lines Approved for Use Under New NIH Guidelines (12/02/2009, National Institutes of Health)

U.S. Approves New Stem Cell Lines for Publicly Funded Research (12/02/2009, HealthDay)

New 'Skin' from Stem Cells (11/19/2009, HealthDay)

Lifelong Exercise Keeps Seniors Young at Heart (11/18/2009, HealthDay)

Stem Cells May Improve Heart Bypass Results (11/17/2009, HealthDay)

Stem Cells May Offer Alternative to Lung Transplants (11/06/2009, HealthDay)

Stem Cells Repair Acute Lung Injury in Mice (11/05/2009, HealthDay)

NIH-Funded Researchers Transform Embryonic Stem Cells Into Human Germ Cells (10/28/2009, National Institute of Child Health and Human Development)

Stem Cell Transplants Stalled Blindness in Rats (10/19/2009, Reuters Health)

Transplant May Treat Metabolic Disorders in Womb (10/16/2009, HealthDay)

Stem Cells Grow Heart Tissue in Lab (10/15/2009, HealthDay)

Stem Cells Being Tested to Fight Pancreatic Cancer (10/14/2009, HealthDay)

Lab Study Makes Strides in Mending Hearts (10/12/2009, HealthDay)

New Way to Make Stem Cells? (10/08/2009, HealthDay)

Stem Cell Research Offers Hope for Colon Cancer Vaccine (10/08/2009, HealthDay)

Stem Cells: Online Resource (2)

2010-01-15T10:01:00.000-08:00

News Sources

Biotechnology Industry Organization Up-to-date news on stem cells, cloning, and regenerative medicine.

EurekAlert! A science news site provided by the American Association for the Advancement of Sciences.

Forum on Science Ethics & Policy Links to informational, legislative, editorial, and other sites discussing stem cell research.

National Geographic: The Power to Divide Feature story on the science and politics of stem cell research.

Nature: Focus on Stem Cells A selection of articles and features on stem cells, including the specially commissioned Stem Cells Insight.

NewScientist: Cloning and Stem Cells A monthly update on stem cell technology.

The New York Times: Stem Cells A free collection of articles about stem cells published in The New York Times.

NOVA scienceNOW: Stem Cells Update From the PBS series (originally aired April 19, 2005). Includes dispatches on the politics of stem cells, the cloning process, and related science news.

PBS: Miracle Cell This episode of the series Innovation explores the use of stem cells for regenerative therapies. (Originally aired April 13, 2004)

Scientific American Provides news coverage on stem cell research. See The Stem Cell Challenge, a feature article from the June 2004 issue.

SciTech Daily Review Offers a range of news articles, features, and commentaries about science and technology topics.

Stem Cell Research News An online newsletter that features stories about stem cells of all types.

Stem Cells The International Journal of Cell Differentiation and Proliferation.

Time: The Stem Cell Debate Stories and resource links on stem cell news.

The Washington Post: Stem Cell Research A collection of articles on stem cell research. (Free registration required to search for other articles within the previous two weeks; fee required to search for older archived articles.)

News Web Logs

The Niche A blog hosted by Nature Reports Stem Cells that offers a forum for debate and commentary on stem cell research and its wider implications for ethics, policy, business, and medicine.

The Stem Cell Blog Discussion on the science, ethics, business, and politics of stem cell research.

Stem Cell News Abstracts of current news articles regarding stem cells, with links to sources.

Stem Cell Research Progress Blog—Information About Stem Cells Discussion of stem cell news and events.

Professional Associations

American Association for the Advancement of Science (AAAS) The AAAS policy brief on stem cell research.

American Medical Association (AMA) Provides information on the basics of stem cell research and highlights relevant policy developments.

American Society for Cell Biology (ASCB) Facilitates the exchange of scientific knowledge about cell biology by disseminating research and training students and young investigators.

American Society for Reproductive Medicine (ASRM) FAQs about cloning, stem cell research, and ASRM's position on the issues.

Association of American Medical Colleges (AAMC) Discussion of stem cell research.

Federation of American Societies for Experimental Biology (FASEB) Seeks to help biomedical and life scientists improve people's health, well-being, and productivity.

The International Cord Blood Society (ICBS) Dedicated to the advancement of stem cell research with an emphasis on umbilical cord blood stem cells.

International Society for Stem Cell Research (ISSCR) Works to exchange information on stem cells, encourage research involving stem cells, and educate the public in all areas of stem cell research and application.

Related Federal Government Sites

FDAA's Center for Biologics Evaluation and Research Determines whether stem cells are safe to use in clinical trials.

Health Resources and Services Administration's The C.W. Bill Young Cell Transplantation Program Provides information and support to patients who need a potentially life-saving bone marrow transplant or umbilicial cord blood transplant.

NIDDK Investigator's Guide to Human Embryonic Stem Cell Research Provides information for researchers supported by the National Institute of Diabetes & Digestive & Kidney Diseases.

NIH Bioethics Resources on the Web Contains a broad collage of annotated web links that provide background information and various positions on issues in bioethics.

The President's Council on Bioethics Advises the President on ethical issues related to advances in biomedical science and technology, including stem cells.

Research Programs at Universities and Institutions

Harvard Stem Cell Institute Supports research into all aspects of stem cell biology, with special emphasis on those areas with the greatest potential for improving human health.

McGowan Institute for Regenerative Medicine Established for University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center scientists and clinical faculty working to develop tissue engineering, cellular therapies, biosurgery, and artificial and biohybrid organ devices.

National Human Neural Stem Cell Resource Provides neural stem cells harvested from the post-natal, post-mortem, human brain to the research community for stem cell research.

The New York Stem Cell Foundation (NYSCF) Offers grants, fellowships, and symposia for stem cell researchers; educates the public about stem cells; and establishes collaborative stem cell research facilities.

Pittsburgh Development Center of Magee-Womens Research Institute Explores the molecular biology of cell function, including the potential of stem cells for treating human disease.

Sloan-Kettering Institute Part of the Memorial Sloan-Kettering Cancer Center, the world's oldest and largest private institution devoted to patient care, education, and research into cancer.

Stanford University School of Medicine/Institute for Cancer/Stem Cell Biology and Medicine Explains Stanford's involvement and perspective on stem cell issues, with links to related sites.

Texas A&M University Mesenchymal Stem Cell Distribution Prepares and distributes well-characterized marrow stromal cells (MSCs) derived from adult human and rodent bone marrow using standardized protocols.

Texas Heart Institute Stem Cell Center Dedicated to the study of adult stem cells and their role in treating cardiovascular disease, including clinical trials (in human patients), as well as many preclinical studies (in the laboratory) using stem cells.

University of California, San Francisco/Developmental and Stem Cell Biology Program Highlights of UCSF human embryonic stem cell research.

University of Massachusetts: International Stem Cell Registry The Potential of Human Pluripotent Stem Cells

University of Minnesota: Stem Cell Institute Works to enhance understanding of stem cells' potential to improve human and animal health.

University of Wisconsin/Embryonic Stem Cell Research Scientists at UW-Madison were the first to successfully isolate and culture human embryonic stem cells.

State Initiatives for Stem Cell Research

California Institute for Regenerative Medicine (CIRM) Makes grants and provides loans for stem cell research, research facilities, and other research opportunities at California universities and research institutions.

Connecticut Stem Cell Research Program Provides grants-in-aid for conducting embryonic or human adult stem cell research.

Illinois Regenerative Medicine Institute (IRMI) Awards grants to Illinois medical research facilities to fund stem cell research involving adult, cord blood, and embryonic stem cells.

Maryland Technology Development Corporation (TEDCO) Provides emerging technology companies and university researchers with funding and specialized technical assistance.

New Jersey Stem Cell Research Program Awards individual research and core facility grants to support human embryonic stem cell research.

New York Stem Cell Science (NYSTEM) Supports basic, applied, translational or other research and development activities that will advance scientific discoveries in fields related to stem cell biology.

Ohio Center for Stem Cell and Regenerative Medicine A multi-institutional center (Case Western Reserve University, University Hospitals Case Medical Center, the Cleveland Clinic, Athersys, Inc., and Ohio State University). Provides basic and clinical research programs, biomedical and tissue engineering programs, and the development and administration of new therapies.

Stem Cell Research State Laws The National Conference of State Legislatures provides an overview of the state laws and pending legislation on stem cell research.

Interstate Alliance on Stem Cell Research (IASCR) Fosters effective interstate collaboration, helps states develop research programs, and promotes efficient and responsible use of public funds.

Stem Cells: Online Resource (1)

2010-01-15T10:00:00.000-08:00

Online Resources

The links included here may connect you to other Internet sites that operate independently of the NIH. The NIH is not responsible for the availability or content of other sites. Permission to reproduce information at other sites may be required. The NIH does not endorse, warrant, or guarantee the information, services, or products described or offered at these external sites.

Annotated Bibliography

Cellular Therapy: Potential Treatment for Heart Disease, Food and Drug Administration (FDA), 2004.
Despite advances in treatment, ischemic heart disease and congestive heart failure are major causes of death in the United States. Cell therapies for treating these diseases are of interest to medical researchers. This resource outlines the issues surrounding clinical studies of human stem cells and the FDA's role in ensuring safe studies.

Stem Cell Therapy for Heart Patients, National Public Radio Talk of the Nation Audio, April 2004.
This one-hour audio program discusses injecting stem cells into heart patients to improve blood flow. This new treatment for heart disease uses the patient's own bone marrow to restore heart function.

Reaping the Benefits of Genomic and Proteomic Research: Intellectual Property Rights, Innovation, and Public Health, Committee on Intellectual Property Rights in Genomic and Protein Research and Innovation, National Research Council, 2005.
The National Academy of Sciences reports on the granting and licensing of intellectual property rights on discoveries relating to genetics and proteomics and the effects of these practices on research and innovation.

Stem Cells and the Future of Regenerative Medicine, by Commission on Life Sciences, 2002.
Stem Cells and the Future of Regenerative Medicine summarizes what we know about adult and embryonic stem cells. It also provides an overview of the moral and ethical problems that arise from the use of embryonic stem cells, compares the likely impact of public and private research funding on progress in the field, and discusses approaches to appropriate research oversight. Based on the insights of leading scientists, ethicists, and other authorities, the authors make recommendations regarding the use of existing stem cell lines versus new lines in research, the important role of the federal Related Federal Government Sites in this field of research, and other fundamental issues impacting potential stem cell-based therapies.

Unlocking the Promise of Stem Cells, Harvard Stem Cell Institute, March 2004.
View an interactive videoconference in which University researchers discuss the Harvard Stem Cell Institute, created to move cutting-edge research on embryonic stem cells from the lab to the clinic.

Beyond Therapy: Biotechnology and the Pursuit of Happiness, Report from Former President Bush's Council on Bioethics, 2003.
Can biotechnology satisfy our human desires—for better children, superior performance, ageless bodies, and happy souls? This report from the President's Council on Bioethics says these possibilities present us with profound ethical challenges and choices. Not declaring "findings," but holding an inquiry—inviting us all to think and debate—the President's Council sought the ideas of dozens of celebrated scientists, thinkers and writers, including such Council members as Francis Fukuyama, Charles Krauthammer, Michael Sandel, and James Q. Wilson, as well as witnesses Steven Pinker, Daniel Schacter, Lawrence Diller, Steven Austad, and S. Jay Olshansky.

Directories and Databases

MEDLINEplus—Stem Cells/Stem Cell Transplantation A consumer health database that includes news, health resources, clinical trials, and more.

Nature Biotechnology Directory A directory of companies engaged in biotechnology research.

PubMed Contains references to literature from more than 4,300 life sciences journals.

Stem Cell Database A joint project of Princeton University and the University of Pennsylvania that contains data on the molecular phenotype of hematopoietic stem cells.

Disease-Specific Organizations and Advocacy Groups

Adult Stem Cell Research Network An internet-based project of The Cell Therapy Foundation designed to be a well-maintained and reliable source of information for the public regarding adult stem cell research, as well as to be a community of practice and collaboration among fellow researchers.

Alliance for Aging Research This citizen advocacy organization works to improve the health and independence of Americans as they age.

ALS Association (ALSA) Dedicated to the fight against Amyotrophic Lateral Sclerosis (also known as Lou Gehrig's Disease), ALSA offers this primer on stem cells.

American Cancer Society Offers information on stem cell transplantation for children with leukemia.

Arthritis Foundation Discusses whether a stem cell transplant cures arthritis.

Christopher and Dana Reeve Foundation Funds research on treatments for central nervous system disorders and works to improve the lives of people with disabilities through a grants program, paralysis resource center, and advocacy efforts.

Coalition for the Advancement of Medical Research Advocates the advancement of breakthrough research and technologies in regenerative medicine, including stem cell research and somatic cell nuclear transfer.

Juvenile Diabetes Research Foundation Using stem cells to restore normal blood sugar, plus information on recent research and policy.

Lymphoma Information Network A page on bone marrow and stem cell transplants.

March of Dimes Aims to give babies a fighting chance against such threats to their health as prematurity, birth defects, and low birthweight.

Research!America Frequently Asked Questions (FAQs) on stem cell research.

Michael J. Fox Foundation for Parkinson's Research Dedicated to ensuring the development of a cure for Parkinson's disease through an aggressively funded research agenda.

National Health Council (NHC) Works to promote the health of all people by advancing the voluntary health movement.

National Marrow Donor Program® (NMDP) Maintains an international registry of donors for all sources of blood stem cells used in transplantation: bone marrow, peripheral blood, and umbilical cord blood.

Spinal Cord Injury Information Network Links to articles about stem cells, particularly for spinal cord injuries.

Educational Resources

Stem Book A collection of invited, original, peer-reviewed chapters on stem cell biology written by top researchers in the field at the Harvard Stem Cell Institute and worldwide. Stem Book is aimed at stem cell and non-specialist researchers.

ExploreStemCells A UK resource for the general public that discusses the use of stem cells in medical treatments and therapies.

How Embryonic Stem Cell Lines Are Made An online animation from the Dolan DNA Learning Center, Cold Spring Harbor Laboratory. (Requires Adobe Flash Player)

Human Embryonic Stem Cells An animated tutorial for the general public. (Requires Adobe Flash Player)

The National Academies Publications on stem cells, including Understanding Stem Cells: An Overview of the Science and Issues from the National Academies (2006), Guidelines for Human Embryonic Stem Cell Research (2005), Stem Cells and the Future of Regenerative Medicine (2002), and Scientific and Medical Aspects of Human Reproductive Cloning (2002).

NIH-Supported Science Education Partnership Award (SEPA) Projects

Cellular Universe: The Promise of Stem Cells Educates visitors about advances in cell biology and stem cells so they can make more informed health-related decisions, explore new career options, and better understand the role of scientific research in healthcare.

If a Starfish Can Grow a New Arm, Why Can't I? This Pittsburgh Tissue Engineering Initiative (PTEI) educational program informs middle school students, their teachers, and the general public about tissue engineering and its applications.

Regenerative Medicine Partnership in Education Duquesne University's SEPA project offers planetarium shows, movies, and educational Web tools.

NWABR Stem Cell Curriculum This 6-lesson unit explores the scientific and ethical issues involved in stem cell research. It features hands-on, engaging classroom activities that have been showcased in the journal American Biology Teacher.

Life Science Learning Center The University of Rochester educational program provide a general overview of stem cell biology and an introduction to stem cell research. Extended Version Stem Cell Biology module features a series of 10 short lessons on stem cells with hands-on lab activities and animated slide show. The Short Version” of the Stem Cell Biology module can be completed in two 40-minute class periods. Instructions for setting up the lab activity are included in the teacher guide.

A Stem Cell Story This 15-minute movie from EuroStemCell introduces the world of stem cell research and is available as a video podcast.

Stem cells: Cells with Potential Part of the San Francisco Exploratorium's Microscope Imaging Station.

Stem Cells: Engage A high school teaching resource on stem cell research developed in Canada.

Stem Cells in the Spotlight and Cloning In Focus The Genetic Science Learning Center at the University of Utah presents these outreach education programs for high school and undergraduate students and teachers.

Stem Cell ResourcesSM A resource for the educational community on stem cells.

Tissue Engineering Planetarium Show Online movies about the body's ability to heal itself.

Tissues of Life: Stem Cells An interactive comic explaining where stem cells are found in the body and how they are gathered.

World Stem Cell Map Reflects national policy and whether public funds may be used to pursue stem cell research using IVF embryos donated from fertility clinics.

International Research

Australian Stem Cell Centre Unites leading Australian academic researchers with the biotechnology industry to develop innovative therapeutic products.

Canadian Institutes of Health Research (CIHR) CIHR is the major federal agency responsible for funding health research in Canada. The site offers links to stem cell research guidelines, the Stem Cell Oversight Committee, and funding opportunities.

Centre for Stem Cell Biology (UK) Part of the University of Sheffield.

EuroStemCell Eleven academic institutes and enterprises from eight European countries compare stem cell information and evaluate their therapeutic potential.

The Goldyne Savad Institute of Gene Therapy (Hadassah University Hospital, Israel) Information on the Institute's Research Group on human embryonic stem cells.

Health Insite (Australia) List of stem cell resources.

Human Embryonic Stem Cell Research in China A September 2002 Report from the U.S. Embassy, Beijing.

Institute for Stem Cell Research (UK) Includes links to its research groups and collaborative initiatives.

Institute of Human Genetics at the University of Newcastle upon Tyne Outlines their current research projects.

The International Stem Cell Forum (ISCF) Encourages international collaboration and funding support for stem cell research.

Kyoto University (Japan) Home page for the University's Stem Cell Research Center

RIKEN Center for Developmental Biology (Japan) Home page for the Center's Laboratory for Stem Cell Biology

Scottish Stem Cell Network A multidisciplinary forum of scientists and clinicians advancing stem cell biology into identifiable health benefits.

Stem Cell Society Singapore Serving the stem cell research community of Singapore.

Stem Cells: Therapies for the Future? (EU) News and information about this 2001 European Union conference.

Tekes—National Technology Agency of Finland Tekes is the main public funding organization for research and development in Finland.

UK Medical Research Council Information on the MRC's stem cell initiative.

The UK Stem Cell Bank Information about stem cell lines available from the UK bank.

UK National Stem Cell Network Promotes research activities and events at the national level to speed the translation of basic stem cell research into therapeutic applications in the control of degenerative diseases.

Review Articles on Embryonic Stem Cell Research (2)

2010-01-15T09:59:00.001-08:00

51.

Regulation of 'stemness' and stem cell differentiation by microRNAs.

Sartipy P, Olsson B, Hyllner J, Synnergren J.

IDrugs. 2009 Aug;12(8):492-6. Review.PMID: 19629883 [PubMed - indexed for MEDLINE]Related articles

52.

Erythropoiesis: model systems, molecular regulators, and developmental programs.

Tsiftsoglou AS, Vizirianakis IS, Strouboulis J.

IUBMB Life. 2009 Aug;61(8):800-30. Review.PMID: 19621348 [PubMed - indexed for MEDLINE]Related articles

53.

Adult neurogenesis and the olfactory system.

Whitman MC, Greer CA.

Prog Neurobiol. 2009 Oct;89(2):162-75. Epub 2009 Jul 15. Review.PMID: 19615423 [PubMed - indexed for MEDLINE]Related articles

54.

Hypoxia and pluripotency in embryonic and embryonal carcinoma stem cell biology.

Silván U, Díez-Torre A, Arluzea J, Andrade R, Silió M, Aréchaga J.

Differentiation. 2009 Sep-Oct;78(2-3):159-68. Epub 2009 Jul 14. Review.PMID: 19604622 [PubMed - indexed for MEDLINE]Related articles

55.

Epigenetic dynamics of stem cells and cell lineage commitment: digging Waddington's canal.

Hemberger M, Dean W, Reik W.

Nat Rev Mol Cell Biol. 2009 Aug;10(8):526-37. Epub 2009 Jul 15. Review.PMID: 19603040 [PubMed - indexed for MEDLINE]Related articles

56.

Making cortex in a dish: in vitro corticopoiesis from embryonic stem cells.

Gaspard N, Gaillard A, Vanderhaeghen P.

Cell Cycle. 2009 Aug 15;8(16):2491-6. Epub 2009 Aug 15. Review.PMID: 19597331 [PubMed - indexed for MEDLINE]Related articles

57.

Role of bioinspired polymers in determination of pluripotent stem cell fate.

Abraham S, Eroshenko N, Rao RR.

Regen Med. 2009 Jul;4(4):561-78. Review.PMID: 19580405 [PubMed - indexed for MEDLINE]Related articles

58.

Wnt signalling and cancer stem cells.

Espada J, Calvo MB, Díaz-Prado S, Medina V.

Clin Transl Oncol. 2009 Jul;11(7):411-27. Review.PMID: 19574199 [PubMed - indexed for MEDLINE]Related articles

59.

Stem cell pluripotency: a cellular trait that depends on transcription factors, chromatin state and a checkpoint deficient cell cycle.

Boheler KR.

J Cell Physiol. 2009 Oct;221(1):10-7. Review.PMID: 19562686 [PubMed - indexed for MEDLINE]Related articles

60.

Neural stem cells in the developing and adult brains.

Qu Q, Shi Y.

J Cell Physiol. 2009 Oct;221(1):5-9. Review.PMID: 19562676 [PubMed - indexed for MEDLINE]Related articles

61.

The glial nature of embryonic and adult neural stem cells.

Kriegstein A, Alvarez-Buylla A.

Annu Rev Neurosci. 2009;32:149-84. Review.PMID: 19555289 [PubMed - indexed for MEDLINE]Related articles

62.

Pluripotent stem cells and other technologies will eventually open the door for straightforward gene targeting in the rat.

Voigt B, Serikawa T.

Dis Model Mech. 2009 Jul-Aug;2(7-8):341-3. Review.PMID: 19553695 [PubMed - indexed for MEDLINE]Related articles Free article

63.

The molecular mechanism of induced pluripotency: a two-stage switch.

Scheper W, Copray S.

Stem Cell Rev. 2009 Sep;5(3):204-23. Epub 2009 Jun 24. Review.PMID: 19551525 [PubMed - indexed for MEDLINE]Related articles

64.

The high mobility group protein HMGA2: a co-regulator of chromatin structure and pluripotency in stem cells?

Pfannkuche K, Summer H, Li O, Hescheler J, Dröge P.

Stem Cell Rev. 2009 Sep;5(3):224-30. Epub 2009 Jun 24. Review.PMID: 19551524 [PubMed - indexed for MEDLINE]Related articles

65.

Induced pluripotent stem cells and the stability of the differentiated state.

Colman A, Dreesen O.

EMBO Rep. 2009 Jul;10(7):714-21. Epub 2009 Jun 19. Review.PMID: 19543232 [PubMed - indexed for MEDLINE]Related articles

66.

Transplantation tolerance in an age of induced pluripotency.

Fairchild PJ.

Curr Opin Organ Transplant. 2009 Aug;14(4):321-5. Review.PMID: 19542888 [PubMed - indexed for MEDLINE]Related articles

67.

The transcriptional foundation of pluripotency.

Chambers I, Tomlinson SR.

Development. 2009 Jul;136(14):2311-22. Review.PMID: 19542351 [PubMed - indexed for MEDLINE]Related articles Free article

68.

When stem cells meet immunoregulation.

Tasso R, Pennesi G.

Int Immunopharmacol. 2009 May;9(5):596-8. Epub 2009 Jan 29. Review.PMID: 19539568 [PubMed - indexed for MEDLINE]Related articles

69.

Making a firm decision: multifaceted regulation of cell fate in the early mouse embryo.

Zernicka-Goetz M, Morris SA, Bruce AW.

Nat Rev Genet. 2009 Jul;10(7):467-77. Review.PMID: 19536196 [PubMed - indexed for MEDLINE]Related articles

70.

Gene regulatory networks in embryonic stem cells and brain development.

Ghosh D, Yan X, Tian Q.

Birth Defects Res C Embryo Today. 2009 Jun;87(2):182-91. Review.PMID: 19530135 [PubMed - indexed for MEDLINE]Related articles

71.

The relationship between pluripotency and mitochondrial DNA proliferation during early embryo development and embryonic stem cell differentiation.

Facucho-Oliveira JM, St John JC.

Stem Cell Rev. 2009 Jun;5(2):140-58. Epub 2009 Apr 3. Review.PMID: 19521804 [PubMed - indexed for MEDLINE]Related articles

72.

An evaluation regarding the current situation of stem cell studies in Turkey.

Arda B, Aciduman A.

Stem Cell Rev. 2009 Jun;5(2):130-4. Epub 2009 May 12. Review.PMID: 19521803 [PubMed - indexed for MEDLINE]Related articles

73.

Policy interoperability in stem cell research: demystifying harmonization.

Isasi RM.

Stem Cell Rev. 2009 Jun;5(2):108-15. Epub 2009 Mar 31. Review.PMID: 19521802 [PubMed - indexed for MEDLINE]Related articles

74.

Diversity in public views toward stem cell sources and policies.

Einsiedel E, Premji S, Geransar R, Orton NC, Thavaratnam T, Bennett LK.

Stem Cell Rev. 2009 Jun;5(2):102-7. Epub 2009 Apr 22. Review.PMID: 19521801 [PubMed - indexed for MEDLINE]Related articles

75.

Emerging ethical, legal and social issues associated with stem cell research & and the current role of the moral status of the embryo.

Zarzeczny A, Caulfield T.

Stem Cell Rev. 2009 Jun;5(2):96-101. Epub 2009 May 14. Review.PMID: 19521800 [PubMed - indexed for MEDLINE]Related articles

76.

The stem cell research environment: a patchwork of patchworks.

Caulfield T, Zarzeczny A, McCormick J, Bubela T, Critchley C, Einsiedel E, Galipeau J, Harmon S, Huynh M, Hyun I, Illes J, Isasi R, Joly Y, Laurie G, Lomax G, Longstaff H, McDonald M, Murdoch C, Ogbogu U, Owen-Smith J, Pattinson S, Premji S, von Tigerstrom B, Winickoff DE.

Stem Cell Rev. 2009 Jun;5(2):82-8. Epub 2009 May 27. Review.PMID: 19521798 [PubMed - indexed for MEDLINE]Related articles

77.

New perspectives in human stem cell therapeutic research.

Trounson A.

BMC Med. 2009 Jun 11;7:29. Review.PMID: 19519878 [PubMed - indexed for MEDLINE]Related articles Free article

78.

GABA's control of stem and cancer cell proliferation in adult neural and peripheral niches.

Young SZ, Bordey A.

Physiology (Bethesda). 2009 Jun;24:171-85. Review.PMID: 19509127 [PubMed - indexed for MEDLINE]Related articles

79.

Large-scale mouse mutagenesis.

Cartwright EJ.

Methods Mol Biol. 2009;561:275-83. Review.PMID: 19504078 [PubMed - indexed for MEDLINE]Related articles

80.

Naive and primed pluripotent states.

Nichols J, Smith A.

Cell Stem Cell. 2009 Jun 5;4(6):487-92. Review.PMID: 19497275 [PubMed - indexed for MEDLINE]Related articles

81.

Embryonic stem cell microRNAs: defining factors in induced pluripotent (iPS) and cancer (CSC) stem cells?

Gunaratne PH.

Curr Stem Cell Res Ther. 2009 Sep;4(3):168-77. Review.PMID: 19492978 [PubMed - indexed for MEDLINE]Related articles

82.

Adult stem cells and mammalian epimorphic regeneration-insights from studying annual renewal of deer antlers.

Li C, Yang F, Sheppard A.

Curr Stem Cell Res Ther. 2009 Sep;4(3):237-51. Review.PMID: 19492976 [PubMed - indexed for MEDLINE]Related articles

83.

Hematopoietic cell development in the zebrafish embryo.

Bertrand JY, Traver D.

Curr Opin Hematol. 2009 Jul;16(4):243-8. Review.PMID: 19491671 [PubMed - indexed for MEDLINE]Related articles

84.

Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease.

Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, Nieto MA.

J Clin Invest. 2009 Jun;119(6):1438-49. doi: 10.1172/JCI38019. Epub 2009 Jun 1. Review.PMID: 19487820 [PubMed - indexed for MEDLINE]Related articles Free article

85.

The basics of epithelial-mesenchymal transition.

Kalluri R, Weinberg RA.

J Clin Invest. 2009 Jun;119(6):1420-8. doi: 10.1172/JCI39104. Review.PMID: 19487818 [PubMed - indexed for MEDLINE]Related articles Free article

86.

EMT: when epithelial cells decide to become mesenchymal-like cells.

Kalluri R.

J Clin Invest. 2009 Jun;119(6):1417-9. doi: 10.1172/JCI39675. Review.PMID: 19487817 [PubMed - indexed for MEDLINE]Related articles Free article

87.

Regulation of stem cell pluripotency and differentiation involves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs.

Kashyap V, Rezende NC, Scotland KB, Shaffer SM, Persson JL, Gudas LJ, Mongan NP.

Stem Cells Dev. 2009 Sep;18(7):1093-108. Review.PMID: 19480567 [PubMed - indexed for MEDLINE]Related articles

88.

MicroRNA and stem cell regulation.

Wang Y, Russell I, Chen C.

Curr Opin Mol Ther. 2009 Jun;11(3):292-8. Review.PMID: 19479662 [PubMed - indexed for MEDLINE]Related articles

89.

Role of ubiquitin ligases in neural stem and progenitor cells.

Naujokat C.

Arch Immunol Ther Exp (Warsz). 2009 May-Jun;57(3):177-88. Epub 2009 May 29. Review.PMID: 19479207 [PubMed - indexed for MEDLINE]Related articles

90.

Pericytes. Morphofunction, interactions and pathology in a quiescent and activated mesenchymal cell niche.

Díaz-Flores L, Gutiérrez R, Madrid JF, Varela H, Valladares F, Acosta E, Martín-Vasallo P, Díaz-Flores L Jr.

Histol Histopathol. 2009 Jul;24(7):909-69. Review.PMID: 19475537 [PubMed - indexed for MEDLINE]Related articles

91.

Genetic regulation of proliferation/differentiation characteristics of neural progenitor cells in the developing neocortex.

Mitsuhashi T, Takahashi T.

Brain Dev. 2009 Aug;31(7):553-7. Epub 2009 May 22. Review.PMID: 19464833 [PubMed - indexed for MEDLINE]Related articles

92.

Stem cells for epidermal melanocytes--a challenge for students of dermatopathology.

Cramer SF.

Am J Dermatopathol. 2009 Jun;31(4):331-41. Review.PMID: 19461236 [PubMed - indexed for MEDLINE]Related articles

93.

Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation.

Snykers S, Henkens T, De Rop E, Vinken M, Fraczek J, De Kock J, De Prins E, Geerts A, Rogiers V, Vanhaecke T.

J Hepatol. 2009 Jul;51(1):187-211. Epub 2009 Apr 2. Review.PMID: 19457566 [PubMed - indexed for MEDLINE]Related articles

94.

Amniotic stem cells for cellular cardiomyoplasty: promises and premises.

Walther G, Gekas J, Bertrand OF.

Catheter Cardiovasc Interv. 2009 Jun 1;73(7):917-24. Review.PMID: 19455667 [PubMed - indexed for MEDLINE]Related articles

95.

Changing nuclear landscape and unique PML structures during early epigenetic transitions of human embryonic stem cells.

Butler JT, Hall LL, Smith KP, Lawrence JB.

J Cell Biochem. 2009 Jul 1;107(4):609-21. Review.PMID: 19449340 [PubMed - indexed for MEDLINE]Related articles

96.

Prospects for pluripotent stem cell-derived cardiomyocytes in cardiac cell therapy and as disease models.

Freund C, Mummery CL.

J Cell Biochem. 2009 Jul 1;107(4):592-9. Review.PMID: 19449339 [PubMed - indexed for MEDLINE]Related articles

97.

Adipose-derived stem cells for tissue repair and regeneration: ten years of research and a literature review.

Mizuno H.

J Nippon Med Sch. 2009 Apr;76(2):56-66. Review.PMID: 19443990 [PubMed - indexed for MEDLINE]Related articles Free article

98.

Pluripotent stem cells.

Verfaillie C.

Transfus Clin Biol. 2009 May;16(2):65-9. Epub 2009 May 12. Review.PMID: 19442552 [PubMed - indexed for MEDLINE]Related articles

99.

Stem cell research and therapy for liver disease.

Navarro-Alvarez N, Soto-Gutierrez A, Kobayashi N.

Curr Stem Cell Res Ther. 2009 May;4(2):141-6. Review.PMID: 19442198 [PubMed - indexed for MEDLINE]Related articles

100.

Adult stem cells as an alternative source of multipotential (pluripotential) cells in regenerative medicine.

Kuçi S, Kuçi Z, Latifi-Pupovci H, Niethammer D, Handgretinger R, Schumm M, Bruchelt G, Bader P, Klingebiel T.

Curr Stem Cell Res Ther. 2009 May;4(2):107-17. Review.PMID: 19442195 [PubMed - indexed for MEDLINE]Related articles

Review Articles on Embryonic Stem Cell Research (1)

2010-01-15T09:58:00.000-08:00

Cardiac fibroblast: the renaissance cell.

Souders CA, Bowers SL, Baudino TA.

Circ Res. 2009 Dec 4;105(12):1164-76. Review.PMID: 19959782 [PubMed - indexed for MEDLINE]Related articles

The use of SC1 (Pluripotin) to support mESC self-renewal in the absence of LIF.

Xiong W, Gao Y, Cheng X, Martin C, Wu D, Yao S, Kim MJ, Liu Y.

J Vis Exp. 2009 Nov 18;(33). pii: 1550. doi: 10.3791/1550.PMID: 19924098 [PubMed - indexed for MEDLINE]Related articles Free article

Generation of induced pluripotent stem cells by reprogramming mouse embryonic fibroblasts with a four transcription factor, doxycycline inducible lentiviral transduction system.

Hamilton B, Feng Q, Ye M, Welstead GG.

J Vis Exp. 2009 Nov 13;(33). pii: 1447. doi: 10.3791/1447.PMID: 19915522 [PubMed - indexed for MEDLINE]Related articles Free article

Multipotent skin-derived precursors: from biology to clinical translation.

Hunt DP, Jahoda C, Chandran S.

Curr Opin Biotechnol. 2009 Oct;20(5):522-30. Epub 2009 Nov 5. Review.PMID: 19896826 [PubMed - indexed for MEDLINE]Related articles

Skeletal morphogenesis during embryonic development.

Yang Y.

Crit Rev Eukaryot Gene Expr. 2009;19(3):197-218. Review.PMID: 19883365 [PubMed - indexed for MEDLINE]Related articles

Genome modification in human embryonic stem cells.

Tenzen T, Zembowicz F, Cowan CA.

J Cell Physiol. 2010 Feb;222(2):278-81. Review.PMID: 19877154 [PubMed - indexed for MEDLINE]Related articles

Isolation and derivation of mouse embryonic germinal cells.

Moreno-Ortiz H, Esteban-Perez C, Badran W, Kent-First M.

J Vis Exp. 2009 Oct 22;(32). pii: 1635. doi: 10.3791/1635.PMID: 19851276 [PubMed - indexed for MEDLINE]Related articles Free article

The role of Runx1/AML1 and Evi-1 in the regulation of hematopoietic stem cells.

Kumano K, Kurokawa M.

J Cell Physiol. 2010 Feb;222(2):282-5. Review.PMID: 19847803 [PubMed - indexed for MEDLINE]Related articles

Molecular events in germ cell tumours: linking chromosome-12 gain, acquisition of pluripotency and response to cisplatin.

Korkola JE, Houldsworth J, Bosl GJ, Chaganti RS.

BJU Int. 2009 Nov;104(9 Pt B):1334-8. Review.PMID: 19840009 [PubMed - indexed for MEDLINE]Related articles

10.

Potential application of adult stem cells in retinal repair--challenge for regenerative medicine.

Machalińska A, Baumert B, Kuprjanowicz L, Wiszniewska B, Karczewicz D, Machaliński B.

Curr Eye Res. 2009 Sep;34(9):748-60. Review.PMID: 19839868 [PubMed - indexed for MEDLINE]Related articles

11.

Gene silencing in human embryonic stem cells by RNA interference.

Rassouli FB, Matin MM.

Biochem Biophys Res Commun. 2009 Dec 25;390(4):1106-10. Epub 2009 Oct 13. Review.PMID: 19833094 [PubMed - indexed for MEDLINE]Related articles

12.

Auxin gradients trigger de novo formation of stem cells during somatic embryogenesis.

Su YH, Zhang XS.

Plant Signal Behav. 2009 Jul;4(7):574-6. Epub 2009 Jul 15. Review.PMID: 19820347 [PubMed - indexed for MEDLINE]Related articles Free article

13.

Gene expression in stem cells.

Liang Y, Russell I, Walworth C, Chen C.

Crit Rev Eukaryot Gene Expr. 2009;19(4):289-300. Review.PMID: 19817706 [PubMed - indexed for MEDLINE]Related articles

14.

Regulation of stem cell pluripotency and neural differentiation by lysophospholipids.

Pitson SM, Pébay A.

Neurosignals. 2009;17(4):242-54. Epub 2009 Sep 30. Review.PMID: 19816061 [PubMed - indexed for MEDLINE]Related articles

15.

Signals involved in neural differentiation of human embryonic stem cells.

Denham M, Dottori M.

Neurosignals. 2009;17(4):234-41. Epub 2009 Sep 30. Review.PMID: 19816060 [PubMed - indexed for MEDLINE]Related articles

16.

Mesenchymal stem cells for bone repair and metabolic bone diseases.

Undale AH, Westendorf JJ, Yaszemski MJ, Khosla S.

Mayo Clin Proc. 2009 Oct;84(10):893-902. Review.PMID: 19797778 [PubMed - indexed for MEDLINE]Related articles

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Complex and context dependent regulation of hematopoiesis by TGF-beta superfamily signaling.

Söderberg SS, Karlsson G, Karlsson S.

Ann N Y Acad Sci. 2009 Sep;1176:55-69. Review.PMID: 19796233 [PubMed - indexed for MEDLINE]Related articles

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The migration of autonomic precursor cells in the embryo.

Kulesa PM, Lefcort F, Kasemeier-Kulesa JC.

Auton Neurosci. 2009 Nov 17;151(1):3-9. Epub 2009 Sep 23. Review.PMID: 19783486 [PubMed - indexed for MEDLINE]Related articles

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Making a neuron: Cdk5 in embryonic and adult neurogenesis.

Jessberger S, Gage FH, Eisch AJ, Lagace DC.

Trends Neurosci. 2009 Nov;32(11):575-82. Epub 2009 Sep 24. Review.PMID: 19782409 [PubMed - indexed for MEDLINE]Related articles

20.

Stem cells and somatic cells: reprogramming and plasticity.

Estrov Z.

Clin Lymphoma Myeloma. 2009;9 Suppl 3:S319-28. Review.PMID: 19778860 [PubMed - indexed for MEDLINE]Related articles

21.

Derivation of insulin-producing cells from human embryonic stem cells.

Van Hoof D, D'Amour KA, German MS.

Stem Cell Res. 2009 Sep-Nov;3(2-3):73-87. Epub 2009 Aug 26. Review.PMID: 19766074 [PubMed - indexed for MEDLINE]Related articles

22.

Skeletal muscle stem cells in developmental versus regenerative myogenesis.

Tajbakhsh S.

J Intern Med. 2009 Oct;266(4):372-89. Review.PMID: 19765181 [PubMed - indexed for MEDLINE]Related articles

23.

Induced pluripotent stem cells in regenerative medicine: an argument for continued research on human embryonic stem cells.

Lee H, Park J, Forget BG, Gaines P.

Regen Med. 2009 Sep;4(5):759-69. Review.PMID: 19761400 [PubMed - indexed for MEDLINE]Related articles

24.

Engineering microenvironments for embryonic stem cell differentiation to cardiomyocytes.

Horton RE, Millman JR, Colton CK, Auguste DT.

Regen Med. 2009 Sep;4(5):721-32. Review.PMID: 19761397 [PubMed - indexed for MEDLINE]Related articles

25.

Molecular regulation of vertebrate retina cell fate.

Andreazzoli M.

Birth Defects Res C Embryo Today. 2009 Sep;87(3):284-95. Review.PMID: 19750521 [PubMed - indexed for MEDLINE]Related articles

26.

Progenitor cell therapy for heart disease.

Gonzales C, Pedrazzini T.

Exp Cell Res. 2009 Nov 1;315(18):3077-85. Epub 2009 Sep 10. Review.PMID: 19747911 [PubMed - indexed for MEDLINE]Related articles

27.

Induced pluripotent stem cells from swine (Sus scrofa): why they may prove to be important.

Roberts RM, Telugu BP, Ezashi T.

Cell Cycle. 2009 Oct 1;8(19):3078-81. Epub 2009 Oct 21. Review.PMID: 19738434 [PubMed - indexed for MEDLINE]Related articles

28.

Chromosomal spread preparation of human embryonic stem cells for karyotyping.

Campos PB, Sartore RC, Abdalla SN, Rehen SK.

J Vis Exp. 2009 Sep 4;(31). pii: 1512. doi: 10.3791/1512.PMID: 19734841 [PubMed - indexed for MEDLINE]Related articles Free article

29.

Stem cells, stress, metabolism and cancer: a drama in two Octs.

Kang J, Shakya A, Tantin D.

Trends Biochem Sci. 2009 Oct;34(10):491-9. Epub 2009 Sep 4. Review.PMID: 19733480 [PubMed - indexed for MEDLINE]Related articles

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Cancer stem cell genomics: the quest for early markers of malignant progression.

Okamoto OK.

Expert Rev Mol Diagn. 2009 Sep;9(6):545-54. Review.PMID: 19732002 [PubMed - indexed for MEDLINE]Related articles

31.

Molecular and cellular determinants for generating ES-cell derived dopamine neurons for cell therapy.

Pruszak J, Isacson O.

Adv Exp Med Biol. 2009;651:112-23. Review.PMID: 19731556 [PubMed - indexed for MEDLINE]Related articles

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Notch signalling and haematopoietic stem cell formation during embryogenesis.

Gering M, Patient R.

J Cell Physiol. 2010 Jan;222(1):11-6. Review.PMID: 19725072 [PubMed - indexed for MEDLINE]Related articles

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Martin-Rendon E, Snowden JA, Watt SM.

Transfus Med. 2009 Aug;19(4):159-71. Review.PMID: 19706133 [PubMed - indexed for MEDLINE]Related articles

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Nuclear receptor regulation of stemness and stem cell differentiation.

Jeong Y, Mangelsdorf DJ.

Exp Mol Med. 2009 Aug 31;41(8):525-37. Review.PMID: 19696553 [PubMed - indexed for MEDLINE]Related articles Free article

35.

Cardiac applications for human pluripotent stem cells.

Shiba Y, Hauch KD, Laflamme MA.

Curr Pharm Des. 2009;15(24):2791-806. Review.PMID: 19689350 [PubMed - indexed for MEDLINE]Related articles

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The role of Krüppel-like factors in the reprogramming of somatic cells to induced pluripotent stem cells.

Nandan MO, Yang VW.

Histol Histopathol. 2009 Oct;24(10):1343-55. Review.PMID: 19688699 [PubMed - indexed for MEDLINE]Related articles Free article

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In vivo fate tracing studies of mammalian taste cell progenitors.

Thirumangalathu S, Barlow LA.

Ann N Y Acad Sci. 2009 Jul;1170:34-8. Review.PMID: 19686103 [PubMed - indexed for MEDLINE]Related articles Free article

38.

Cancer stem cells: the other face of Janus.

Mittal S, Mifflin R, Powell DW.

Am J Med Sci. 2009 Aug;338(2):107-12. Review.PMID: 19680015 [PubMed - indexed for MEDLINE]Related articles

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Emerging concepts in neural stem cell research: autologous repair and cell-based disease modelling.

Koch P, Kokaia Z, Lindvall O, Brüstle O.

Lancet Neurol. 2009 Sep;8(9):819-29. Review.PMID: 19679274 [PubMed - indexed for MEDLINE]Related articles

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New complexity in differentiating stem cells toward hepatic and pancreatic fates.

Huppert SS, Magnuson MA.

Sci Signal. 2009 Aug 11;2(83):pe50. Review.PMID: 19671927 [PubMed - indexed for MEDLINE]Related articles

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Deciphering the stem cell machinery as a basis for understanding the molecular mechanism underlying reprogramming.

Bosnali M, Münst B, Thier M, Edenhofer F.

Cell Mol Life Sci. 2009 Nov;66(21):3403-20. Epub 2009 Aug 7. Review.PMID: 19662495 [PubMed - indexed for MEDLINE]Related articles Free article

42.

Toward clinical therapies using hematopoietic cells derived from human pluripotent stem cells.

Kaufman DS.

Blood. 2009 Oct 22;114(17):3513-23. Epub 2009 Aug 3. Review.PMID: 19652198 [PubMed - indexed for MEDLINE]Related articles

43.

Stem/progenitor cells in liver injury repair and regeneration.

Zhao Q, Ren H, Zhu D, Han Z.

Biol Cell. 2009 Jul 31;101(10):557-71. Review.PMID: 19642968 [PubMed - indexed for MEDLINE]Related articles

44.

Dissection of gene regulatory networks in embryonic stem cells by means of high-throughput sequencing.

Beisel C, Paro R.

Biol Chem. 2009 Nov;390(11):1139-44. Review.PMID: 19642873 [PubMed - indexed for MEDLINE]Related articles

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Current progress and prospects of induced pluripotent stem cells.

Chen L, Liu L.

Sci China C Life Sci. 2009 Jul;52(7):622-36. Epub 2009 Jul 30. Review.PMID: 19641867 [PubMed - indexed for MEDLINE]Related articles

46.

Generation of pancreatic islet cells from human embryonic stem cells.

Zhang D, Jiang W, Shi Y, Deng H.

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Human parthenogenetic embryonic stem cells: one potential resource for cell therapy.

Hao J, Zhu W, Sheng C, Yu Y, Zhou Q.

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Embryonic stem cell models in vascular biology.

Li X, Claesson-Welsh L.

J Thromb Haemost. 2009 Jul;7 Suppl 1:53-6. Review.PMID: 19630768 [PubMed - indexed for MEDLINE]Related articles

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Progenitor cell trafficking in the vascular wall.

Hristov M, Weber C.

J Thromb Haemost. 2009 Jul;7 Suppl 1:31-4. Review.PMID: 19630763 [PubMed - indexed for MEDLINE]Related articles

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Stem cells, pluripotency and nuclear reprogramming.

Jaenisch R.

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