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How to make a Mammoth Crisp(r)

Did you see the autopsy of the incredibly well-preserved woolly mammoth last week??

I particularly enjoyed watching a group of scientists totally nerding out over a 40,000 year old frozen corpse – especially that one guy who was so darned determined to find a nice bit of mammoth poo! *Spoiler alert* – he finds some!

There wasn’t the same enthusiastic jumping-up-and-down excitement that followed the recent meteor landing, but I suppose there was more risk of a terrible mess.

During the programme, they mentioned a technique called ‘CRISPR’ (pronounced as we always aim to have our chips (or ‘fries’ to Americans) … ‘crisper‘) as a way to change or edit elephant DNA to be more similar to woolly mammoth DNA. And it’s not just mammoth cloners that are excited about this technique – the whole of genetic and molecular science is silently and carefully jumping up and down about it.

That’s because it’s pretty cool. It’s exciting because it replaces older technologies that take a lot longer, costs a lot more and are much more complicated. CRISPR can therefore potentially reduce the method of gene editing down from years to months at a fraction of the price. Although the actual practicalities of getting it to work are pretty fiddly, the general protocol is actually relatively straight forward.

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So what is CRISPR?

CRISPR is not just a spelling mistake, it’s an acronym. It stands for ‘Clustered Regularly Interspaced Short Palindromic Repeats.’ Or put in simpler terms: short repeated sequences of DNA that read the same forwards and backwards, which tend to group together and have similar sized spaces between them. But ‘SRSODTRTSFABWTTGTAHSSSBT’ isn’t so pithy.

CRISPR DNA is found in bacteria and acts as their main form of defence against foreign DNA, such as from a virus. CRISPR RNA (CrRNA) locates and attaches to foreign DNA with a complementary sequence of nucleotides (see Express Yourself! for more on this). Identifying the intruder by CrRNA signals to a special enzyme, called ‘Cas9’ that can then cut (or ‘cleave’) the foreign DNA, leaving it inactive and harmless. This kind of enzyme is known as an endonuclease. The CRISPR/Cas9 system acts similarly to our immune system, in that it can remember previous infections in order to protect against future ones. The DNA sequence of an offending virus is assimilated into the CRISPR DNA so that it may be quickly identified, attacked and neutralised if it ever has the tenacity to attack again!

How is that going to bring back woolly mammoths? And why is it such a big deal?

Well, because molecular geneticists have been able to hijack this system so that it can cleave and edit the bits of DNA that they are interested in. The CRISPR/Cas9 system can be isolated from bacteria, and expressed in other cell types – such as elephant cells, or human brain cells. This means that scientists can fiddle about with different genes, and see how that changes the way cells function.

For example, imagine that a cell is expressing a gene associated with a particular disease. Stopping that gene from working – or ‘silencing’ it, may be an effective therapy or cure for that disease. To do this, scientists engineer a portion of the CRISPR DNA so that it recognises the gene they want to attack – in the same way that CRISPR would recognise previous viruses. This means that crRNA will track down the target gene, signal to Cas9 to cut it and stop it from working. Voila! The gene is silenced.

Or imagine it this way – a sniffer dog has been given the scent of a criminal they need to track down.

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The scent acts as its guide to find the target, just as the DNA sequence from an interesting gene acts as a guide for the CRISPR system. The dog can then bring the criminal down, and CRISPR cuts the gene.

So how about gene editing rather than silencing? To do this, scientists hijack a different cellularfunction called ‘homologous recombination.’ Homologous recombination means that a piece of DNA that is broken can repair itself with a near-identical fragment of DNA that acts as a template. In order to edit genes, new DNA ‘templates’ are manufactured in the lab with the required edit, which are then added to the cell. That means that once CRISPR/Cas9 cuts the DNA, there will lots of near-identical templates available for cells to use to repair themselves – therefore editing their gene in the process.

If we take this back to the sniffer dog example, the homologous recombination process would be like putting a skin graft on a nasty bite the dog inflicts on the criminal – it’s not exactly the same as the original skin, but it’s close enough for a repair. Perhaps while they’re at it, the police add an electronic ankle tag so they can easily find the ‘edited’ criminal – this can also be done in cells by adding a fluorescent tag to the CRISPR/Cas9 system so it’s easy to see which cells have or haven’t been edited down a microscope.

And that’s how they might genetically engineer a mammoth! Or treat particular diseases! Or prevent inherited genetic conditions! The range of possibilities is vast, but the technology is still very much in its infancy and needs a lot of fiddling with.

However – the quick, easy and reliable editing of the human genome as a method for treating and curing disease is currently molecular genetics’ enthusiastic hunt for a woolly mammoth turd. We just need to keep on digging.

 

The Biocheminist

 

For more details about CRISPR, see:

http://www.molim.uni-erlangen.de/downloads/Teaching/Hauptseminar_Aspekte/Manuskripre_WS12_13/Tag14_Crisp/2010_Horvath_Science_Review.pdf

http://www.nature.com/nbt/journal/v31/n3/full/nbt.2508.html

http://www.annualreviews.org/doi/abs/10.1146/annurev-genet-110410-132430?journalCode=genet

https://www.addgene.org/crispr/reference/history/

https://www.addgene.org/CRISPR/guide/

http://www.scientificamerican.com/article/fact-or-fiction-mammoths-can-be-brought-back-from-extinction/

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Growing cells – it’s a culture thing

Growing cells – known as Cell Culture – is a fundamental process carried out in most biochemistry research labs. Having a never-ending supply of cells available is a valuable resource for researchers. It allows us to manipulate cells and investigate the effects of new drugs in a way that would be impossible, expensive and unethical to do in animal models or in people. They also provide a consistent and plentiful source of material to perform lots of experiments in a relatively short period of time.

There are hundreds of different types of cells, referred to as ‘cell lines’, which come from different parts of the body, different species, and are created in different ways.

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Some neuronal cells growing in a dish

Broadly, there are 2 categories of cell line:

  1. Primary

These cells are taken directly from a piece of tissue, and have a finite lifespan. They will not continue to grow and divide, so are used in short-term experiments.

  1. Continuous

Continuous cells have originated from a piece of tissue, but they have been transformed in the lab so that they continue to grow and divide indefinitely. These are often referred to as ‘immortalised’ cells. The most famous and most common cell line is known as HeLa, which originated from a biopsy of an extremely aggressive case of cervical cancer. HeLa cells are a particular oddity as they appear to have transformed themselves without any manipulation in the lab. HeLa cells have a complicated and controversial history relating to medical and research ethics – to find out more about them, I would highly recommend reading ‘The Immortal Life of Henrietta Lacks’ by Rebecca Skloot (don’t worry, it’s not too sciencey!).

So what do you need to grow cells?

All cell lines are different and may have specific needs, but the basics are the same. Cells are grown in a nutrient-rich liquid referred to as ‘media.’ Media helps stabilise cells and provides the essential nutrients required for cells to grow.

In loose terms, growing cells isn’t too dissimilar to growing a human baby – it’s a case of food in and waste out, and some care in between to make sure they don’t get sick. It’s also beneficial to avoid dropping them on the floor. Media therefore commonly contains the following:

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Growing cells just like growing babies….kinda

Glucose: Provides energy to the cells,

Glutamine: An amino acid that acts as an extra energy source

Phenol Red: A pH indicator, which changes colour if the acidity of a solution changes. Cell culture media is commonly a reddish-pink colour because of the phenol red, but if the culture becomes too acidic, perhaps by cell overgrowth, infection or an accumulation of waste, then the media will turn a gross yellowish colour so it is easy to see when something is wrong. Media needs to be removed and replaced regularly, as the cells will use up energy and consequently produce waste, which is toxic to the cells if it builds up.

Antibiotics: To help prevent any unwanted infections.

Serum: The remaining component of blood after clotting and the removal of any remaining blood cells. The most common serum used in cell culture is fetal bovine serum (from cow fetuses), referred to as ‘FBS,’ and is a by-product of slaughterhouses for the meat industry. Serum is essential in cell culture because it provides all of the components normally present in the body that helps cells to grow and survive, such as proteins, carbohydrates, hormones and vitamins.

Sadly, there is no additive to correct researcher clumsiness.

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Cell culture in action using media containing phenol red

But it’s still not quite as straightforward as feeding and cleaning!

Cells have to be cultured in special sterile conditions – because the cells are no longer growing in a complicated system made up of hundreds of different cell types and a functional immune system, they have no protection against infection. The addition of antibiotics to the media helps protect against bacterial infection, but they are no substitute for proper sterile technique!

Sterile technique involves using a special cabinet (or hood) that has a particular flow of air. Air is sucked into the cabinet and passed through a filter to get rid of any nasties before reaching the area containing the cells. Used air is extracted from the cabinet and disposed of elsewhere. Everything that enters the hood is sprayed with ethanol, and all of the equipment, such as pipettes and tubes, are always certified as sterile by the manufacturer and are only ever used once to prevent any potential contamination.

Cells must also be grown in special incubators that carefully regulate their environment – the majority of cells will grow best at 37˚C (body temperature – what a coincidence!), with some humidity and 5% carbon dioxide in the air, which helps maintain the correct pH.

What happens once you have a batch of cells happily growing?

They grow some more!

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Happy cells are growing & dividing cells

Continuous cells will carry on dividing and growing – they will run out of space and nutrients, so will eventually poison themselves and starve if left to their own devices. This means that cells need to be regularly ‘split’ (officially called ‘passaging’) – this simply means that the cells in one flask or dish will be split up into several other flasks or dishes to continue to grow with more space and more nutrients. This method means that cells can quickly be bulked up into huge numbers and can then be prepared and used for various experiments.

I’ve spent the majority of my fledgling research career doing cell culture, so I’m bound to be biased, but I think it’s pretty awesome.

 

If you have any questions about cell culture, feel free to ask in the comments section below, and let me know if you have any other biochemistry or neuroscience questions you’d like answered! You can also follow me on Twitter @TheBiocheminist

The Biocheminist

Biochemistry is just like cooking… but try not to eat it

When I started my PhD, I was told that if you could follow the recipe in a cookbook, you could successfully carry out most experiments (success being measured here by a lack of spilling/breaking/wasting/ruining/blowing up anything, rather than by the experiment actually working AND giving you the result you hoped for).This is because experiments normally follow a specific protocol, which is fundamentally the same as following a recipe. However, the more I’ve worked in a lab, the more I’ve seen the similarities with a kitchen… So here are some of the regular day-to-day kitchen things used commonly in the lab:

Cling film & Tin foil

Both cling film and tin foil are used on a daily basis – although special lab versions are available, normal supermarket brand versions are used a lot. Cling film is used for pretty much the same thing in labs as in the kitchen – to wrap things up for storage, to stop contamination, spillages, and evaporation. Tin foil is used to keep light out of things that may degrade in light – for example, when working with fluorescent tags and antibodies, the experiment will be kept under tin foil to prevent fading of the fluorescent signal.

Fridge freezer

The success of many an experiment is down to proper storage of your samples, and everything needs a different storage temperature. While the lab has fancy freezers set at -80˚C for RNA and long term sample storage, as well as liquid nitrogen dewars for cryopreserving cells at around -200˚C, there are also regular old fridge freezers. Fridges are set to +4˚C and are used for short term storage of DNA, some antibodies and various chemicals and reagents. Freezers are set to -20˚C, and are used to store all kinds of things, including protein samples, DNA and antibodies.

Microwave

There’s not much to say about this one! In the lab the microwave is used to heat up and melt things, although very rarely would those things ever be considered edible.

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Milk

Marvel skimmed milk powder in particular is a laboratory favourite. It is most commonly used to make up a ‘blocking buffer’ for western blots – this is typically 5% milk powder in a saline/detergent solution (see ‘Western What’s??’ and its comments section!)

Milkshake

Milkshake brings all the mice to the yard – I mean – helps mice learn associations. Sweetened or condensed milk and milkshakes are used as rewards in mouse and rat learning experiments. For example, a mouse may learn to press a lever in response to a flashing light because they are given a drop of delicious milkshake when they do what they are supposed to do. The milkshake is positive reinforcement – exactly the same as treating my husband to coffee & cake when he goes shopping with me without complaining. I hear from colleagues that strawberry milkshake is a mouse favourite (and also a husband favourite).

Yeast

Just like the stuff used in bread and beer! Although for lab use, it comes from a more controlled and regulated source than the dried variety from the shops. Yeast is a single-cell organism – and its simplicity has allowed the creation of various models that can be used to study fundamental processes in cells that are required for life, for example how proteins interact with each other and how the cell cycle works. It has been particularly useful because it is so easy to grow and manipulate.

Nail varnish

Not really a kitchen accessory, but I’m sure someone will have painted their nails in a kitchen at some point. Specifically the clear, quick drying variety is preferred! A common way of looking at cells under a microscope is to grow the cells on a circle of glass called a ‘coverslip.’ Then when there are enough cells, the coverslip is placed upside down onto a glass microscope slide, so that the cells lie between the two layers of glass. Clear nail varnish is then painted around the coverslip to seal it onto the microscope slide and to stop the sample from drying out.

I’m sure there must be more household things used regularly in labs – especially with scientific ingenuity and tightened budgets! I like to think I’m pretty good at cooking, and I can follow a protocol pretty darn well! However, most important of all, it’s of utmost importance to make sure there’s always enough milk, both at home and in the lab, as running out in either place can really ruin my day!

The Biocheminist

N.B. Posts will now be appearing fortnightly rather than weekly, for the sake of the posts on here and for the sake of my experiments in the lab!

Express Yourself!

There is a fundamental part of cell biology that I haven’t posted about yet, but I have skimmed over it briefly in previous posts here & here. I must admit, the link to Madonna is tenuous (at best), but I will be writing about one of the many ways we all express ourselves. In contrast to the expression of feelings that Madge sung about in the 80’s, this form of expression is not under your conscious control; the fundamental process described in this post is: Gene Expression!

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Madonna, expressing herself.

What is it?

Gene expression describes the process by which your cells can convert your DNA (deoxyribonucleic  acid) into other molecules or products that have particular jobs/functions within the cell. The most common example of this is the conversion of DNA into proteins, which can then go off and carry out different jobs around the cell. Different cell types (e.g. a heart cell, a blood cell or a neuron) exist because they read different parts of your DNA, and consequently create different proteins that carry out different jobs and functions (see the ‘instruction manual’ metaphor in this previous post!).

So how do your cells convert DNA into proteins?

This is done by a combination of two processes called Transcription and Translation.

But before I get into that, here are the basics of DNA:

DNA is a double stranded molecule. It can be compared to the two sides of a zip (or zipper) that fit together in the middle. Instead of the teeth that fit together on the zip, DNA has individual nucleotides (also called ‘bases’). These are simpler molecules that are the building blocks of DNA. There are four different nucleotides:

Adenine (referred to as A)

Cytosine (referred to as C)

Guanine (referred to as G)

Thymine (referred to as T)

In the DNA ‘zip,’ an A on one strand is always paired up with a T on the other strand, and a C is always paired up with a G. These pairings (perhaps unsurprisingly) make up Base Pairs.

Transcription

The first process that occurs in gene expression is transcription, and happens in the cell nucleus.

This is the way that DNA is converted (or Transcribed!) into RNA. RNA (ribonucleic acid) is the single stranded equivalent of DNA. Like DNA, it is also made up of four different nucleotides; however while it has the A, C and G nucleotides, it has Uracil (U) in the place of T. The two strands of DNA are first separated by an enzyme called DNA helicase, so that the nucleotides are no longer paired and are exposed ready to be transcribed. Another enzyme, RNA polymerase, then starts ‘reading’ the nucleotides along the open strand of DNA and creates a complementary strand of RNA. This piece of RNA is referred to as the transcript.

Not all of your DNA is transcribed all at once – only the genes that are needed for that cell at that time are transcribed, so the single strands of RNA tend to be much shorter than the double strands of DNA. There are actually many different types of RNA that exist within cells, and they can carry out different functions. However, the form of RNA that goes on to help create proteins is called messenger RNA, or ‘mRNA’ for short.

transcription

Translation

Translation is the process where mRNA is converted (or ‘Translated’!) into a protein. After its creation, mRNA moves into the cytoplasm of the cell, so this is where translation occurs. To understand how translation works, you need to know these five things:

  1. 1. Proteins are made up of chains of amino acids.
  2. 2. Different combinations of amino acids make up different proteins
  3. 3. A set of three nucleotides in a row will ‘code’ for an amino acid. This sequence of three nucleotides is called a ‘Codon.’
  4. 4. Different combinations of nucleotides will code for different amino acids
  5. 5. Therefore the sequence of nucleotides on a strand of RNA provide the instructions for a particular chain of amino acids, which in turn create a particular protein. A different sequence of nucleotides on different strands of RNA will therefore provide the instructions for a different chain of RNA, resulting in a different protein.

translation 1

So how does translation occur?

A molecule called a Ribosome attaches to the mRNA – ribosomes are like tiny protein-processing factories, and guide the translation process. The ribosome attaches to the first codon – this guides another form of RNA, tRNA (transfer RNA), to that codon. tRNA forms a link between nucleotides and amino acids. On one side it has a codon, and on the other side it has the amino acid that the codon represents or gives the instruction for.

The tRNA needs to have a complimentary codon sequence to the mRNA; if you remember that nucleotides pair together –an mRNA codon made up of CCG would require the tRNA to have a complimentary codon of GGC in order to pair up. This process makes sure the correct amino acids are assembled in the correct order.

The ribosome moves along the mRNA, recruiting tRNA with its attached amino acids as it goes. The amino acids then form a chain, which creates a protein. Once the ribosome has finished moving along the mRNA, the constructed protein is released, and translation is complete!

translation 2

Transcription and translation are complicated mechanisms, but hopefully I made that clear enough to follow! However, they form a fundamental part of gene expression, which underlies how our bodies (including the best bit – the brain (obviously!) work and grow.

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Labyrinth, thinking about gene expression

And although Madonna may have missed the mark, Labyrinth’s more recent version of ‘Express Yourself’ might be little more accurate when he says ‘Being myself is something I do well’ – Yup, thanks to biochemistry and cell biology, we all express ourselves, and generally we do it pretty damn well!

The Biocheminist

PC Arrrggghh!

PCR (see what I did there?) isn’t as terrifying as the title of this post suggests – although it has been known to induce screams of frustration in poor hard working students and researchers!

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So what is PCR?

The ‘Polymerase Chain Reaction’ (don’t worry – the meaning of this will become completely clear!) is one of the most commonly used lab techniques. Briefly, it is a technique to ‘bulk up’ (or Amplify) a certain piece of DNA that you are interested in from a sample of mixed bits of DNA. This makes it easier to find the interesting piece among all the boring bits you aren’t interested in.

There are several reasons why you might want to do this, but most frequently it is used to check whether a certain piece of DNA is present in your sample.

Why would that be useful?

  • You would want to do this if you tried to change the DNA in a cell line or animal model and you need to check if it worked.
  • Alternatively, PCR can be used to see if there are any natural mutations between, say, the DNA from ‘healthy’ people and the DNA from a group of people with a particular disease
  • Along the same lines as above, it can be used to test for genetic diseases (where the cause of the disease is known to be in the DNA and is passed down through families)
  • Out of the research lab and hospitals, it is also the technique used for paternity testing (as appears frequently on The Jeremy Kyle Show etc) and in forensic science (g. as seen in CSI) – in these cases, two DNA samples are compared for their similarity to each other, or in the case of forensic science, it can also create a much larger sample for testing from an initially very small trace of DNA left at a crime scene.

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Finding what you want in the DN-hAystack! (LOL)

But what actually is the polymerase chain reaction?

 

It’s a tricky one to explain as there are several stages, so first I’ll note the two important things you need to start with, followed by the process itself. A word of warning – it’s one of those things where looking at the pictures really helps!

You need to:

  1. Collect your DNA sample

This might be from a cell line, from a lab rat sample or from a sample taken from a patient or volunteer. Typically, the DNA is then ‘extracted’ from the cells (as animal/human samples will consist of cells – which is also where DNA is stored). By extracting the DNA and getting rid of the other bits of the cell, you should get a ‘cleaner’ sample that is less likely to fail during the PCR. This sample is called the ‘DNA template.’

  1. Prepare your primers

DNA is made up by a chain of ‘bases’ (or ‘nucleotides’) – Cytosine, Guanine, Adenine and Thymine (C, G, A and T), which pair together to form a double stranded DNA helix. In order for the PCR technique to amplify the part of DNA you are interested in, you need to tell it what part of the DNA to pay attention to. This is done with ‘primers’ (as they prime the reaction). Primers are short, single stranded chains of bases that are designed to match (‘complement’) the sequence of bases on the interesting region of DNA.

Steps for PCR:

  1. Denaturation

This is basically just separating the two strands of DNA from each other to form single strands. This is so that the primers are able to pair with their complementary sequence on the DNA strand. Denaturation is done by briefly heating the DNA to 94-98˚C.

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  1. Annealing

The temperature is dropped to 50-65˚C to allow the primers to pair (‘anneal’) with the DNA

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  1. Extension

An enzyme called DNA Polymerasehence the ‘P’ in ‘PCR!) recognises the primer-DNA pair, and recruits spare bases/nucleotides from the surrounding area (these are added by the researcher, along with the DNA polymerase).

The DNA polymerase is typically taken from a bacteria called ‘Thermus Aquaticus’ and is referred to as ‘Taq’ – this is used because it can withstand the high temperature used in step 1, whereas polymerase from any other source would break down and stop working.

The polymerase then synthesises a new strand of DNA that matches the original strand of DNA. Primers are designed to match both strands of DNA (as the sequence of the second strand will be reversed compared to the first), so during the extension phase, both the ‘forward’ and ‘reverse’ strands of DNA are synthesised to form a copy of double stranded DNA.

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  1. And repeat!

The previous three stages are repeated between 20-40 times. Each time it is repeated, the amount of DNA is doubled, so that there is an exponential increase in the number of copies of the DNA sequence you are interested in (until the spare nucleotides and DNA polymerase run out).

Now you have loads of a specific sequence of DNA! Yay!

The resulting DNA can be passed through a gel and separated by size in the same manner as the protein described in Western Whats?‘ A difference in size indicates a different nucleotide sequence, and therefore a potential mutation, or mismatch between samples. Large amounts of amplified DNA are also required for other biochemical techniques such as sequencing (which reads the whole sequence of the DNA strand one nucleotide at a time) or for inserting into the DNA of another organism, such as yeast or bacteria (with the purpose of seeing the effect this region of DNA may have on a cell).

So that seems pretty straightforward, right?

Well, yes, PCR is one of those things that can be very easy – but only when it works! Unfortunately, every stage of PCR is very sensitive to disruption, and many different sequences of DNA will need slightly different conditions for the PCR to work. For example, both too much and too little template DNA can completely ruin a PCR, and if the primers are not specific enough to the region of interest, they can pair with the wrong section of your template DNA and cause all kinds of rubbish to be amplified!

It therefore takes an experienced/skilled/lucky researcher to get a perfect PCR first time; otherwise you start to hear those screams….

 

The Biocheminist

 

Did this post make sense to non-scientists? I’d love some feedback on how understandable my posts are and if I’m managing to explain biochemistry and neuroscience to you!

Is there more about PCR you would like to know? Or are there any other lab techniques or neurological diseases you’d like to learn more about? Comment below!

Western Whats??

Western blots.

I do a lot of western blots. They can be a source of outbursts of happiness accompanied by triumphant dancing, or (and currently, most commonly for me) be the cause of crippling despair!

So what’s a western blot?

And how does it have such power to control a poor post-doc’s emotional well-being?

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It is actually a relatively simple method of finding out whether a particular protein is present in the sample you are investigating (for example, a type of cell line, a bit of tissue from the body or a blood sample). It can also tell you whether one sample has more or less of that particular protein than another sample, or the size of a protein you are interested in.

The reason for doing this is to determine whether cells of one type are working in the same manner as cells of another type. For example, a western blot could tell you whether  ‘disease’ cells have more or less of a particular protein, which may make cells act differently (see my previous blog I fiddle about with cells in a dish for a clearer explanation!).

Proteins are made up of different length chains of amino acids, which results in proteins of many different lengths. The longer a protein is, the larger its mass (as it consists of more amino acids), and shorter proteins are smaller. The mass of a protein is measured in ‘kilodaltons’ (kDa). Each sample that is to be investigated is made up of a mixture of hundreds of different proteins. Therefore, to find the one you are looking for, these proteins need to be separated by their size/mass. This is done by using an electric current to push the sample through a gel, which is essentially a kind of mesh.

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In this mesh, small proteins can pass through very quickly, whereas large proteins struggle to move through the gaps and therefore move slower. The proteins will now be separated by their size rather than being in one mixed mess. A protein ‘ladder’ is passed through at the same time, which has particular sized proteins stained with a dye so that you know what size proteins are present at what point down the gel mesh.

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Where’s the blot part?

Well, this isn’t actually the western blot – this is just the necessary preparation for a western blot. This stage is actually called ‘sodium dodecyl sulfate polyacrylamide gel electrophoresis (or SDS-PAGE)’ – but don’t worry about that.

The blot part comes next. So the proteins are trapped in a gel, but gel breaks easily and is very fragile, so the proteins are transferred over  – or blotted – to a fabric ‘membrane’ that is easier to work with. This blotting is achieved by using an electric current to force the movement of proteins out of the mesh onto the membrane, which consists of lots of little pores that trap the proteins and lock them into place.

Once proteins are locked into place on the membrane, antibodies can be used to pick out the specific protein that you are interested in. Each antibody is designed to be specific to a particular protein, and theoretically should only attach to that protein and that protein alone (although the reality is often different!). When the antibody is attached, there are several methods by which it can be detected with specialised equipment. When it is detected, a ‘band’ of protein is made visible on the membrane. The size of the detected band can then be estimated by comparing it to the ladder that has dyed proteins of already known sizes.

If more of the protein you are looking for is in your sample, the band will appear fatter (and thinner if there is less of that protein). If there have been changes to the protein that might affect its size, then the band will appear above (if larger) or below (if smaller) where it is normally found on the blot.

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(A finished western blot – the ladder is on the left, the band of protein is present in the middle of the membrane. Each blob is a different sample, and each sample has a different size blob/band, suggesting different amounts of that protein between them)

The happiness comes from whenever a blot works (and when the results are consistent with what you predicted!). The disappointment stems from the many times they don’t work…this can be due to many variables such as broken equipment, non-specific antibodies, contamination, poor detection, out of date chemicals, bad blotting, old samples, and most recently in our lab, dodgy water! Sometimes they might not work for any other reason than just because the western blot gods are angry that day.

One more thing…Why are they called ‘western’ blots?

Truthfully, there is no real reason. It is a hilarious biochemical in-joke. There was already a similar method of detecting DNA called a ‘Southern blot’ (named after its inventor Prof. Southern). A method of detecting RNA (the single-stranded version of DNA) was subsequently named as ‘Northern blot,’ so it was decided that the equivalent detection of proteins would carry on the theme. An ‘Eastern blot’ for the detection of protein modifications also exists.

 

Feel free to comment and let me know if this made sense, or if there is anything I’ve failed to explain clearly, or if there are more details you would like to know!

 

The Biocheminist

Using Animals for Neuroscience Research

It’s a very touchy subject for some people, and as it was reported last week that the number of animal experiments being carried out has increased this year, I decided I would try to discuss some of the reasons behind using animals in research.

I’m not an expert in animal research, but I have had some experience of carrying out some animal experiments, have worked in the animal labs and work in a department that is actively engaged in using animal models of disease. So, unsurprisingly, I am not against the use of animals in scientific research (cosmetic and household testing is a completely different matter that I won’t be getting into here). However, I recognise, accept and agree that it is an uncomfortable idea. Pair that discomfort with the apparent secrecy surrounding many scientific research labs, and it’s easy to see why animal research, and the people who conduct it, are treated with suspicion.

If they are hiding something, then there must be something awful to hide, right?

From my experience, and from knowing the people who work with animals in research, this is really quite far from the truth. The animals most commonly used in research are, as everyone knows, rats and mice. This is because they are relatively small mammals that are capable of learning, and have some similarities to human biology. The animals in the labs are cared for round the clock by qualified technicians, many of whom used to be veterinary nurses, and a vet is always on call and makes regular visits. On top of this, the use of animals in the UK is under the strictest regulations in the world to ensure the highest level of animal welfare, and regular visits by inspectors ensure that this is the case.

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But, while the animals are very well cared for, they are ultimately used for research purposes, and that’s the uncomfortable bit. Within neuroscience research, this may include deliberate injuries to the brain (carried out under strict and sterile surgical conditions), treatment with new or experimental drugs, or may consist purely of ‘behavioural tests’ – where the animal learns associations between signals and a reward (think Pavlov and his dogs) or where to go in a maze to get some food. In addition, the majority of mice used in research have been altered or bred to have a particular genetic mutation that mimics the mutations found in human diseases. This creates an ‘animal model’ of a disease, so that how a disease progresses over time can be investigated, or new experimental treatments for symptoms can be tested.

Without prior knowledge of the importance and usefulness of these experiments, or the details of how the animals are treated and cared for during the process, it’s easy to think that this is simply cruelty, carried out by evil scientists who are merely satisfying their curiosity.

This opinion may arise from a lack of information, and details from outdated studies. Before writing this post, I took a look on some popular anti-vivisectionist (anti-animal testing) websites to see the other side of the argument. While much of the information given on the websites was incorrect and outdated (particularly with regards to housing conditions, and on one webpage, all the references were at least 14 years old), there is one important argument that I wanted to address.

The most common argument is that it is pointless to use animals because they do not reflect the human condition, so anything that is carried out in mice is unlikely to work in humans and is a waste of time.

Well, there are two sides to this argument.

Yes, obviously humans are different to rats and mice. Their brains are different, and many drugs that have shown promising results in animals have failed in human clinical testing. But not all drugs have. Animal models have allowed us to uncover an abundance of essential information about many neurological diseases that couldn’t be identified in humans, and has pushed forwards our understanding of disease and improved research in the process. They are an essential part of investigating the effects of genetic manipulations, as well as the basic biology of disease and are a starting point for developing future life-saving treatments.

But, that being said, I don’t think there is a single scientist who would say that animal models are perfect.

So why do we have to use animals?

Well, a big reason is the lack of an alternative that can provide us with as much information. While it is being argued that human cell models, including the development of stem cells, are more relevant to medical research (they are human after all), the problem, particularly with the brain and in neuroscience, is one of connectivity. The brain is made up of many different cell types, all of which are responsible for different functions. But they are all connected and they communicate with each other, which means that they can affect and alter each other. This complexity has not been replicated in a petri dish with cells, but it is present in an animal brain. Something that may work in one cell model won’t necessarily work in a different cell model, never mind in a full working brain – whether it is human or not. Even if we could eventually one day grow an entire functioning brain just from cells in the lab, then would it have consciousness? And if was a conscious human brain, then experimenting on it becomes ethically questionable (at the very least!).

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Cells also can’t tell us the behaviour that may result from an experiment – for example, a drug to improve the symptoms of Alzheimer’s disease may have the expected biochemical effects in a cell model, but does it actually improve memory? And are there any side effects? Another drug predicted to treat Parkinson’s disease may not change the cell as anticipated, but would it still give back some movement control?

The usefulness of animal models is frequently discussed within the scientific community, and the general consensus tends to be that although they are not the perfect solution, animal models need to remain an integral part of scientific research until appropriate and accurate alternatives can be developed. Unfortunately, we aren’t there yet. In the meantime, improved communication between scientists and the public may ease some of the tension and suspicion about what goes on in animal labs, and the fear of being targeted or attacked for working in animal research. It may or may not change any minds, but both sides will at least be better informed.

What are your views on scientific animal research? Is there anything you wish you knew more about? I’d love to hear your comments!

 

The Biocheminist

Why do scientists want my DNA?

I came across an article on the BBC news website this week that was discussing the pressure some individuals in Iceland are under to donate samples of their DNA to the private genetic research company deCODE (http://www.bbc.co.uk/news/magazine-27903831). The UK has an equivalent charitable organisation that collects several different types of biological samples from individuals over time; the UK Biobank (links to both deCODE and UK Biobank’s webpages are at the end of this post).

So why do scientists want your DNA, and what’s so special about the Icelandic variety?

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DNA (or deoxyribonucleic acid, if you want to impress your friends) acts like the instruction manual for the cells in your body, and therefore dictates how they behave, grow and function. Different cell types in your body (for example, a skin cell vs. a heart cell) ‘read’ from different ‘chapters’ of this manual, depending on what their job is (e.g. to produce skin pigment vs. rhythmically contract to make a heartbeat).

Keeping with the instruction manual metaphor …

DNA (the manual) is made up of genes (individual instructions). Every individual person has a different version of the manual, due to having slightly different versions of the instructions. The end result is ultimately the same (a human being!), but with a few variations. Some of these variations are obvious – such as hair or eye colour, height or nose shape. Other variations are less obvious, and may make a person more susceptible, or resistant to developing a particular disease or illness.

There may also be a typo in some of the instructions. Sometimes these don’t matter – the instructions can be read anyway and have the same result as if spellcheck had been completed. In other cases, the typo might change the meaning of the instructions, and the cell doesn’t quite work properly, therefore possibly causing a disease. These typos are referred to as ‘Single Nucleotide Polymorphisms’ (or SNPs).

In genetic research, the particular version of an instruction/gene you have is called a ‘Gene Variant.’

It is rarely the case that one particular gene variant causes a disease 100% of the time. It is more common that several different gene variants are ‘associated’ with a disease – that is to say that if you have one or more of those variants, you are more likely to get a particular disease. But, whether you actually do or not depends on many other things, such as your other genes and your diet & lifestyle.

But finding these ‘risk’ gene variants isn’t very straightforward.

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And that’s why geneticists (gene scientists) want your DNA!!

It is now possible to scan the whole of the DNA (speed-read the manual…if you want to keep the metaphor) from an individual relatively quickly, and this method is being used to find the genetic variants that might increase the risk of disease. However, as well as these important risk gene variants, human DNA is full of useless noise! Because a particular variant won’t ‘cause’ a disease 100% of the time, huge numbers of individual’s DNA needs to be read to try and sift out the important stuff from the noise.

So this takes us back to Iceland –

Genetic ‘noise’ can come from mixtures of different ethnicities and backgrounds; different continents typically have different genetic ‘signatures,’ which account for differences in disease rates, as well as more obvious features such as appearance. With immigration and international mobility, it is difficult to find a large population of people with a very similar genetic background to each other that would reduce the genetic noise and make the risk gene variants easier to find. However, Iceland is that population, and that is what makes their DNA particularly useful for research.

So what is the point of finding risk variants if they don’t necessarily cause the disease?

Well, the eventual aim is to identify the combination of risk variants and lifestyle factors that can accurately predict whether someone will develop a particular disease. A recent high profile example of the usefulness of this knowledge is Angelina Jolie’s choice to have a mastectomy to prevent the development of breast cancer because she carried the variant of the BRCA1 gene that increases the likelihood of developing the disease. Such interventions will be more complicated for neuroscience (removing the brain isn’t really an option), but identifying important genes can also focus the research done in labs so that it can be as targeted and specific as possible to increase the chances of finding a cure.

The ultimate (and very cool) goal is to be able to accurately predict the likelihood that any individual will develop a disease based on their genes and lifestyle, and tailor a treatment to their own personal genetic makeup. Although I feel I should point out, that’s a very long way off, and there is a whole minefield of ethical issues about whether people wish to share their DNA, how it will be stored, whether people want to know if they will develop a disease or not….perhaps the subject of another post.

I encourage you to take a look at the UK Biobank website for more information and perhaps even consider getting involved in some research!

 

The Biocheminist

 

www.deCODE.com

www.UKBiobank.ac.uk

 

I fiddle about with cells in a dish

This is the short, succinct, and yet still pretty accurate description of what I do at work! This is the description I roll out at parties following a few glasses of wine, when there is only time for a five minute interaction, and when bumping into distant acquaintances who have only asked me what I do to be polite.

And while it very effectively sums up what I and many others do, the details of those cells and how they came to be in that dish are pretty awesome.

I research Huntington’s Disease – and although this will most likely be the subject of a future post, I’ve put some of the main points that you might be interested in at the end of this post.

So the big question is; what’s the relevance of a bunch of cells in a lab to a complicated, devastating human disease?

Well, a major roadblock to investigating any disease that affects the brain is that it isn’t considered particularly ethical or practical to cut into a live person’s brain and cut out a bit to take a look at.

Damn.

So that’s where fiddling about with cells in a dish come in. The particular cells I work with have been engineered to carry the mutation that causes Huntington’s Disease – making it a cell model of Huntington’s Disease.

A particularly cool bit of this process is that the cells are ‘immortalised’. This is exactly as it sounds – the cells are made immortal so that they continue to grow and divide instead of dying after a week or so. This is done by forcing the cell to follow its internal programs for growth and survival that would normally be turned off.

The advantage of using a cell model is that people like me can investigate what is different between those cells that carry the mutation and those that don’t. The idea behind this is to try to understand the disease on a molecular and cellular level – that means we ask things like; how do they look different? Do they react differently to treatments? Do they have different levels of protein? Are they communicating differently? How can we fix it?

 

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Perhaps more importantly, this never-ending supply of cells means that we can look for something that might prevent the effects of the Huntington’s Disease mutation – or whatever mutation is being investigated –  and this (after many years of work and plenty of failures!) may then sow the seeds for the development of new treatments and cures.

That’s the ultimate goal.

Cell models are a crucial starting point for understanding how individual cells work, or rather stop working, in the context of a disease  – especially when that disease attacks the very inaccessible cells in the brain! The endless supply of cells also gives fantastic flexibility – experiments can be relatively easily repeated and new techniques can be trialled without losing valuable source material – because you can just grow some more!

Compare this with animal work (another future blog post!) – where each individual animal is a precious resource and the numbers used must be kept to a minimum –  and with human studies – where only so many people with the disease exist or have been identified, only a proportion will be willing or able to take part in research programs, and will understandably only consent to so many blood samples and tests.

Of course, cell models are by no means perfect – the brain is incredibly complicated, and a lot of cells in a dish can’t possibly replicate that (but there are people working on it!). But that’s not to say there’s no point in using them – I’m not wasting my time, I swear!! They are a brilliant spring board; a preliminary stage in the investigation of new ideas, and they can be manipulated (i.e. fiddled about with) in ways that aren’t possible in animals or people. Although they aren’t the perfect model and cannot be considered as an exact replication of human disease, cell models are still a crucial and valuable resource in scientific research.

 

The Biocheminist

Please feel free to comment if you want to know any more about cell models in research, or more about Huntington’s Disease!

 

Huntington’s Disease – The basics

  • Huntington’s Disease is caused by a mutation on the (imaginatively named) ‘huntingtin’ gene. It typically starts to develop in mid-life (around 40-50 years old).
  • The child of a person carrying the mutated gene has a 50% chance of inheriting the gene.
  • The gene is ‘dominant’ – which means that anyone who has the mutation will develop the disease in their lifetime. If the gene is not passed down from parent to child, that child will not develop Huntington’s Disease, and they in turn cannot pass it on to their children.
  • The cells in a particular region of the brain – the striatum – no longer function properly and start to die. This is called ‘neurodegeneration.’
  • The striatum controls muscle movement, so losing its function means that people with Huntington’s Disease slowly lose control of their bodies – typically this starts with ‘chorea,’ also described as ‘dance-like movements.’
  • The disease spreads throughout the brain, so sufferers also lose some cognitive abilities such as decision making, problem solving & memory.
  • Although the genetic cause for the disease is known, there is currently no cure.

 

 

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