Improving reproductive cloning

Remember when Dolly the sheep was cloned in 1996?  That was the first cloned mammal and everyone freaked out thinking we would be cloning all our pets and even humans within a few years.  Well, nearly 20 years have passed since then and reproductive cloning is still a very difficult and inefficient procedure.  Most cloning has a 1-5% success rate.  Why is that?  Before we can answer that, we need to understand the procedure for reproductive cloning.

Somatic cell nuclear transfer

Our bodies are made up lots of different types of cells – neurons, skeletal muscle, intestinal cells, immune cells, etc.  Despite the different functions and structures of these cells, all of the cells in one organism have the same genome, the same set of genes.  What makes cells unique is that they express different genes at different times, so different proteins are made.  What this means is that the blueprint (DNA) for making a new organism is right there in every cell in your body.

The normal way of making an embryo is by taking half of the genome from a male (in sperm) and half from a female (in the egg) and combining them during fertilization.  In reproductive cloning, you already have a whole genome from any adult cell.  That nucleus from the adult can be inserted into an oocyte (or egg) that has had its DNA removed (“enucleated”).  The egg is necessary because it has lots of nutrients and signals in it that are important for the first few cell divisions during early development.

This process is shown in the diagram below and is called somatic cell nuclear transfer.  In the example, the adult genome is coming from a fibroblast cell and is transferred into an enucleated oocyte.  This is a way to get stem cells (ntES), which can be used for therapeutic purposes, like making more neurons that can be transplanted into someone with Parkinson’s.  Or you could let the cloned cells grow up into an embryo and then into a cloned organism.

Somatic nuclear cell transfer (from Stembooks.org)

Although it is possible to make cloned organisms using adult donor genomes, the efficiency is much higher when using genomes from embryos.  What happens to the adult genome that prevents it from directing the formation of a new organism?  This problem is addressed in a recent paper by Matoba et al., published in Cell.

Epigenetic changes

Although adult cells should have the same DNA sequences as their embryonic precursors, the genome can be organized differently, which can affect which genes are expressed.  DNA wraps around histone proteins as a way to organize the long DNA chains.  Histones can be modified in such a way that the DNA will wrap around more tightly or more loosely.  For example, if a particular amino acid in histone 3 is trimethylated (three CH3 groups are added), then that makes the DNA pack up closer together, so it is really hard to express those genes.  There are genes that may need to be expressed early on in development, so their histones will be modified to allow for loose packing, but then after they are expressed, they’ll get packed away, so they take up less space.  These kinds of modifications that affect gene expression are called epigenetics.

As an analogy, imagine you have had a child, so you have baby clothes, a crib, car seat and toys in your house.  Once that child grows up, you take all those baby things down to the basement.  You still have them, but you will probably never need to use them again, so you can pack them all up and store them so they are out of the way.  It may be hard to access them again, but they are still there.  So a gene that has been packed away into condensed chromatin is still present in a cell, but it is no longer giving instructions for making proteins, unless something comes along and unpacks it.

You can see now the problem with somatic cell nuclear cloning.  The adult cell already has some DNA packed away, so when the genome is put into an oocyte, it may be impossible to express the genes necessary to direct normal development.  In the paper by Matoba et al., they did indeed find that there are more trimethyl modifications (called H3K9me3) in mouse embryos derived from nuclear transfer than embryos from in vitro fertilization (using a sperm and egg).  These regions were associated with decreased gene expression and compact DNA.

Improving efficiency

Now we know one of the problems, but what can researchers do to improve the efficiency of reproductive cloning?  Somehow they need to decrease H3K9me3 modifications in the donor genome.  They do this two ways:

(1) There are too many methylated histones, so the authors injected an enzyme that removes methyl groups into the one-cell embryos.  The embryos expressed more genes and survived throughout development.  70% of these cloned embryos implanted into a surrogate mouse uterus and 8% survived to adulthood.  Those numbers are higher than before, but still not perfect.

(2) They also tried decreasing expression of the enzymes that put on the methyl groups.  This also improved development, so 50% of the embryos made it to later stages of development, but they did not see how many survived to adulthood.

There is an epigenetic barrier for nuclear transfer from adult cells in mouse oocytes.  Presumably a similar problem is preventing cloning in other organisms as well.  It makes sense that an adult cell would have a different pattern of epigenetic modifications than an embryonic genome.  The authors were able to improve cloning efficiency by decreasing the H3K9me3 modification, but there are probably other histone modifications that are also different in adults.  There is still a long way to go before cloning is a reliable procedure, but at least now we have some explanation of why it is so difficult.

Transgenerational inheritance of fear

A new semester has begun and I have no extra time to update this blog, so just a short entry today.  This paper was just too cool to pass up.  It was published earlier this year in Nature Neuroscience by Dias and Ressler.  They conducted a series of experiments which showed that learned fear can be passed on from generation to generation in the sperm DNA.  That’s Lamarckian evolution for all you evolution nerds out there.  A learned behavior that is inherited genetically -- totally crazy!

The DNA sequence itself isn’t changing, but instead the expression of genes is altered, so different amounts of proteins are being made.  This process is known as epigenetics (which I’ve discussed before with regard to histone modifications).  One way to change DNA expression is by methylating cytosines (the “C” in DNA sequences).  The methyl group (CH3) makes it harder for proteins to bind to the DNA and transcribe the genes into mRNA and subsequently into protein.  The general rule of thumb is: more methylation --> less gene expression, less methylation --> more gene expression.  This is a common way our cells regulate gene expression, and what’s really interesting is that many external influences can affect DNA methylation, like traumatic life experiences, smoking, exercise, environmental toxins, etc. 

Cytosine getting methylated (note the H3C addition on the the molecule on the right)

It is conceivable that DNA methylation patterns can be inherited through generations, so changes in gene expression that affected your great grandparents could still be maintained in your cells.  Most of these types of studies focus on changes that occur to the mom during pregnancy.  For example, lets say that researchers expose a pregnant mouse to a toxin that may affect DNA methylation.  The next generation (the F1 generation) is also being exposed in utero to the same toxin.  The eggs or sperm progenitor cells are also developing in the embryo, so the next next generation (F2) may also be exposed to the toxin.  Thus, if you see DNA methylation changes in the F1 and F2 offspring, this isn’t really so surprising since these cells were all exposed at the same time as the mom (F0 generation).  In order to really prove that DNA methylation patterns can be inherited across generations (transgenerationallly), you need to expose the parents to a stimulus before conception, before the F1 and F2 generation even exist, which is what they did in this paper.

The authors chose to initiate changes to gene expression by conducting odor conditioning in mice.  They paired a particular odor with a mild foot shock and conditioned F0 males to be afraid of the odor.  Then these males mated with naïve females (never exposed to the odor).  The F1 offspring showed excessive fear to the conditioned odor, even though they had never encountered it before.  It was their fathers who had been shocked, not them.  The authors found there were more cells in the olfactory region of their brains that expressed the olfactory receptor for the conditioned odor.  Furthermore, their dad’s sperm and their own sperm were less methylated in the gene that encodes for that particular odor receptor, so the offspring of F1 were also affected.

F0 dad learns fear for odor --> decreased methylation for receptor gene --> F1 offspring inherit methylation changes --> express more of the receptor --> increased sensitivity to odor --> F1 sperm have same decreased methylation --> sensitivity for odor passed on to F2 generation  

Grandpa (F0) had a bad experience with an odor and now his grandkids will be more afraid and sensitive to that odor.  And it’s all genetic.  The authors did a series of experiments to show that it isn’t behaviorally based (grandpa isn’t telling the grandkids about his horrible experience with this smell).  For instance, they took the sperm from the F0 mice and took it to a different mouse facility and did in vitro fertilization, so the father was nowhere near his offspring or the mother.  The F1 mice from in vitro fertilization were just as super sensitive to the odor.

This is nuts!

Here's a good blog post about this paper

Isolation and drug addiction

We all know that adverse, early life experiences can affect normal development and the ability to lead a happy and healthy adult life.  A number of recent studies have shown that rodents which are mistreated as pups have long lasting changes to their gene expression (i.e. epigenetics).  They are more anxious and have a harder time forming new memories.  A paper this week in Neuron builds upon these results, by studying the effects of social isolation on the “reward pathway” in the brain.

Reward pathway

What is a reward pathway?  Deep in the brain is a region known as the ventral tegmental area (VTA), which makes connections to the nucleus accumbens and prefrontal cortex.  When we do something that is naturally good, like eating or sex, the neurons in the VTA release dopamine onto the nucleus accumbens and we interpret that as “feeling good”.  This is our reward for doing something that will help us survive and procreate. 

The Reward Pathway in a brain cross section (from brainfacts.org)

Many drugs of abuse like cocaine, amphetamines and alcohol increase the amount of dopamine signaling in this pathway; this is one reason why drugs produce a “high”.  When this pathway gets overstimulated by increased drug use, the brain will try to compensate by making the pathway less efficient.  This is why drug users feel depressed when not on drugs and why higher and higher concentrations of drugs are necessary to produce the same high feeling.  This is a neurological explanation for drug addiction.  Drug abusers also start to make connections in their lives, and in their brains, between environments (a certain room, certain people, etc) and the feeling of reward.  Getting sober is so difficult because the brain has to unlearn these connections and the reward system has to recover back to its normal level of activity.

Plasticity

Before we talk about the paper, I need to introduce one more concept.  Neurons become activated when channels in their membranes open and positive ions rush in.  They can then pass on this signal to another neuron by releasing neurotransmitters (like dopamine) onto the next neuron.  The activity in the neuron and the amount of transmitter it releases into the synapse can change over time, based on that neuron’s previous experiences.  This is known as synaptic plasticity.  There are short-term changes, like facilitation, and longer-term changes (we’re talking hours and days here).  One of the more famous types of long-term plasticity is called long term potentiation (LTP) and is thought to underlie learning and memory.  When drug users start to become addicted, these types of long term changes to neuronal activity are occurring throughout the reward pathway.

Social isolation and VTA neurons

The experiment begins when young male rats were either housed together in groups of 3 or alone.  The researchers then recorded neuronal activity of VTA neurons under various conditions.  They found that rats that were isolated for more than 3 weeks, specifically during the equivalent of the rats’ early adolescence, can more easily induce LTP in the VTA neurons.  In other words, rats that had no social interactions during a critical period had more sensitive VTA neurons.  That is to say, their reward pathway is primed to be overstimulated, just like during repeated drug use.

What are the behavioral manifestations of having a sensitive reward pathway?

The next set of experiments they did is called conditioned place preference.  The rats were placed in a cage that had two different compartments, with different wall colors and floor textures.  The rats were then injected with amphetamine in one of those particular compartments, so they learned to associate the drug high with that environment.  The rats were then given a choice between the two compartments and inevitably they went to the room that was associated with the drug.  The researchers found that isolated rats had a greater preference for the drug room and developed the preference sooner than the control rats.  Social isolation as an adolescent causes an increased rate of learning an association between drugs and environment.  This could make these rats more vulnerable to drug addiction. 

What about unlearning the drug association?

After the drug testing, the rats were exposed repeatedly to the drug room, but this time they didn’t receive any drugs.  This is called extinction of a memory and it is measured by the rats losing their preference for the former drug compartment.   Socially isolated animals had a significantly slower rate of unlearning the preference.  Their memory was more resistant to extinction.  If their VTA neurons are overly sensitive, then it may be harder to rewrite that connection in the brain between environment and reward.

In the context of drug addiction, these findings are big.  An adverse early adolescence can prime the brain to develop addiction more easily and make it harder to sober up.  If the VTA neurons start firing every time you go through an environment associated with drugs, you’re going to want to take a hit again.  The authors bring up an interesting point that social isolation generally causes a depression of neuronal activity in places like the hippocampus (the site of learning), so maybe the increased activity in the VTA is the way for the brain to maintain some sort of homeostasis – some areas increase, some decrease, but overall the brain may have normal amounts of activity.  This is an interesting way of looking at this problem.  I suspect that social isolation offers little in the way of rewards, so the reward pathway is trying to compensate by getting more sensitive.  It will be interesting to see if there is also a connection with changes in gene expression.  The authors explain how the VTA neurons get overactive, from a cellular point of view, but what actually initiates those changes?  And how can social interactions feed into the biology of the cell? 

The buzz about novelty-seeking

One topic I have always been interested in is how complex behaviors are driven by genetics and molecular biology.  For instance, all the courtship rituals of a male fruit fly are basically encoded by a single gene, and more specifically one small exon (a chunk of DNA within a gene).  A recent paper by Liang et al. which was published in Science, looked at novelty-seeking behavior in honeybees.  The nice thing about this paper is that they started out by finding bees that like to scout for food and nests and then worked backwards to figure out what is different about these bees compared to their boring, steady-as-she-goes sisters.

Novelty-seeking in bees

 There are two types of novelty-seeking behaviors which the authors investigate. 

1) Food scouting: Even when there are plentiful known food sources, some bees (5-25% of foragers) still go out to look for new sources.  The other foraging bees rely on these food scouts, who tell them where the new food source is.  The food scouts are obviously important for a colony, so the bees always know where to get food.

Photo taken by Rebecca Wenk 2010.

2) Nest scouting: When a swarm has left its colony in search of a new location, a few bees (<5%) will search out for the best place to build a new hive.  They will tell the rest where to go and lead them there.

Are nest scouts the same bees as food scouts?

The researchers identified nest scouts in artificial and natural swarms and marked them.  Then they waited to see if these nest scouts would become food scouts as well.  They moved the hives every night to a new location.  The bees that seek new food sources will not be thrown by the new environment and will be able to find new food sources quickly.  Under these circumstances, the authors found there was a trend for the former nest scouts to also be food scouts, though it wasn’t a hard-and-fast rule.

Molecular underpinnings

All worker bees are female and have the same genetic make up— they’re all identical sisters.  What would make some bees more likely to explore novel environments?  To examine this question, the authors collected food scouts.  While the bees slept in their hives, the authors introduced a new feeder in different locations each day.  They collected bees that visited the new feeder twice, when it was in two different locations.  Once they had the food scouts, they performed a microarray to determine differences in gene expression between food scouts and non-scout controls.

If you want to know more about how microarrays work, visit my methods section.  All you really need to know is that a microarray looks at every single gene and determines how much it is expressed.  Since all bees have the exact same genes, the only differences are going to arise by how these genes are expressed (remember: not all genes are expressed at a given time).

The authors found that scouts have differential expression of various neurotransmitter systems.  Neurotransmitters are the signals that are sent from one neuron to another.  They found that scouts had higher expression of genes involved in GABA and glutamate neurotransmission.  In general, GABA is used to inhibit neurons, and glutamate is the standard excitatory neurotransmitter.  GABA and glutamate may control different neuron circuits in the brain.  They also saw a decrease in a receptor for dopamine.  What this implies is that when the brain has increased GABA and glutamate signaling, but decreased dopamine, it makes the bees want to seek out novel environments (or maybe it makes them less afraid to try something new). 

To prove that this hypothesis is correct, the authors took some non-scouts and fed them glutamate.  These non-scouts with extra glutamate were more likely to search for new food.  In other words, they switched the behavior of non-scouts to novelty-seeking by changing the balance of neurotransmitters in their brain.  Unfortunately, this didn’t work when they tried to do the same thing with dopamine.  They inhibited the dopamine receptor, which should also increase food scouting, but it actually made the non-scouts even less likely to scout for food.

It’s cool that the authors did behavioral studies in bees, but I feel like something was missing in this paper.  Okay, neurotransmitter systems are expressed differently, but why?  A paper from last year showed that queen bees have different epigenetic marks than worker bees, so maybe this is what’s going on with the novelty seekers.  Is there some difference in their environment as they develop, which causes epigenetic changes and differential gene expression?  There is a lot more to explore in this topic.